Modified variant bowman birk protease inhibitors

ABSTRACT

The present invention relates to modified variant Bowman Birk Protease Inhibitor proteins (BBPIs) that comprise peptides that bind target proteins, and that are further modified to have greater protease inhibitory activity and/or be produced at greater yields than the unmodified BBPIs. The invention encompasses polynucleotide constructs and expression vectors containing polynucleotide sequences that encode the modified variant BBPIs, the transformed host cells that express and produce the modified variant BBPIs, the modified variant BBPI proteins, the compositions comprising the modified variant BBPIs, and the methods for making and using the modified variant BBPIs in personal care.

1. FIELD OF THE INVENTION

The present invention relates to modified variant Bowman Birk ProteaseInhibitor proteins (BBPIs) that comprise peptides that bind targetproteins, and that are further modified to have greater proteaseinhibitory activity and/or be produced at greater yields than theunmodified BBPIs. The invention encompasses polynucleotide constructsand expression vectors containing polynucleotide sequences that encodethe modified variant BBPIs, the transformed host cells that express andproduce the modified variant BBPIs, the modified variant BBPI proteins,the compositions comprising the modified variant BBPIs, and the methodsfor making and using the modified variant BBPIs in personal care.

2. BACKGROUND OF THE INVENTION

Proteases are involved in a wide variety of biological processes.Disruption of the balance between proteases and protease inhibitors isoften associated with pathologic tissue destruction.

Various studies have focused on the role of proteases in tissue injury,and it is thought that the balance between proteinases and proteinaseinhibitors is a major determinant in maintaining tissue integrity.Serine proteinases from inflammatory cells, including neutrophils, areimplicated in various inflammatory disorders, such as pulmonaryemphysema, arthritis, atopic dermatitis and psoriasis. These and otherinflammatory conditions are often associated with dysregulated levels ofcytokines.

Proteases also degrade the vascular basement membrane and participate inthe remodeling of the extracellular matrix to facilitate cell migrationand invasion to promote tumor angiogenesis. Proteases release angiogenicgrowth factors bound to the extracellular matrix and to generatechemotactically active fragments derived from extracellular matrixcomponents, which in turn exert chemotactic and mitogenic, modulatoryactivates on endothelial cells, smooth muscle cells and fibroblasts toparticipate in angiogenic processes. The list of protein factorsangiogenically active in vivo includes fibroblast growth factors,Angiogenin, Angiopoietin-1, EGF, HGF, NPY, VEGF, TNF-alpha, TGF-beta,PD-ECGF, PDGF, IGF, IL8, and Growth hormone. Risks associated withcurrent cytokine blocking agents include serious infections,anaphylaxis, and lupus-like syndrome. In addition, there is observedloss of clinical benefit after the drugs are stopped, and a smallproportion of patients develop antibodies to the biological agents,which is likely to limit their efficacy with repeated use.

Synthetic and natural protease inhibitors have been shown to inhibittumor promotion in vivo and in vitro. Previous research investigationshave indicated that certain protease inhibitors belonging to a family ofstructurally-related proteins classified as serine protease inhibitorsor SERPINS, are known to inhibit several proteases including trypsin,cathepsin G, thrombin, tissue kallikrein, as well as neutrophilelastase. The SERPINS are extremely effective at preventing/suppressingcarcinogen-induced transformation in vitro and carcinogenesis in animalmodel systems. Systemic delivery of purified protease inhibitors reducesjoint inflammation and cartilage and bone destruction as well.

Topical administration of protease inhibitors finds use in suchconditions as atopic dermatitis, a common form of inflammation of theskin, which may be localized to a few patches or involve large portionsof the body. The depigmenting activity of protease inhibitors and theircapability to prevent ultraviolet-induced pigmentation have beendemonstrated both in vitro and in vivo. Paine et al., Journal ofInvestigative Dermatology 116:587-595 [2001]. Also, protease inhibitorshave been found to help wound healing(http://www.sciencedaily.com/releases/2000/10/001,002071718.htm).Secretory leukocyte protease inhibitor was demonstrated to reverse thetissue destruction and speed the wound healing process when appliedtopically. In addition, serine protease inhibitors can also help toreduce pain in lupus erythematosus patients (See U.S. Pat. No.6,537,968).

Naturally occurring protease inhibitors can be found in a variety offoods such as cereal grains (oats, barley, and maize), Brussels sprouts,onion, beetroot, wheat, finger millet, and peanuts. One source ofinterest is the soybean. The average level in soybeans is around 1.4percent and 0.6 percent for Kunitz and Bowman-Birk respectively, two ofthe most important protease inhibitors. These low levels make itimpractical to isolate the natural protease inhibitor for clinicalapplications.

Thus, there is a need for a method to produce large quantities ofprotease inhibitors that have desired characteristics of proteintherapeutics, and for compositions that effectively deliver the proteaseinhibitor in a usable form.

The compositions and methods according to the invention fulfill some ofthe above needs and in particular offer an advantage in providingprotease inhibitors that specifically target the activity of cytokinesinvolved in pathologic and non-pathologic processes.

3. SUMMARY OF THE INVENTION

The present invention relates to modified variant Bowman Birk ProteaseInhibitor proteins (BBPIs) that comprise peptides that bind targetproteins, and that are further modified to have greater proteaseinhibitory activity and/or be produced at greater yields than theunmodified BBPIs. The invention encompasses polynucleotide constructsand expression vectors containing polynucleotide sequences that encodethe modified variant BBPIs, the transformed host cells that express andproduce the modified variant BBPIs, the modified variant BBPI proteins,the compositions comprising the modified variant BBPIs, and the methodsfor making and using the modified variant BBPIs in personal care.

In one embodiment, the invention provides for an isolated modifiedvariant Bowman Birk Protease Inhibitor (BBPI) that comprises asubstituted amino acid at least at one amino acid position chosen frompositions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50,52, 55, and 65 of the variant BBPI of SEQ ID NO:187, and in which thesecond protease inhibitory loop of the BBPI scaffold is a bindingpeptide. In one embodiment, the BBPI scaffold is chosen from thescaffolds: BBI (SEQ ID NO:13), BBIt (SEQ ID NO:185), BBI-AV (SEQ IDNO:186), BBIt-AV (SEQ ID NO:187), BBIt-VEGK (SEQ ID NO:640), BBIt-VEGT(SEQ ID NO:641), BBIt-VEGKD (SEQ ID NO:642), BBdb (SEQ ID NO:449), BBsb3(SEQ ID NO:450), BBtc (SEQ ID NO:451), BBdb-AV (SEQ ID NO:452), BBsb3-AV(SEQ ID NO:453) and BBtc-AV (SEQ ID NO:454). In another embodiment, thebinding peptide is chosen from a VEGF binding peptide, an FGF-5 bindingpeptide, a TGFβ binding peptide and a TNFα binding peptide. TheVEGF-binding peptide in turn is chosen from ACYNLYGWTC (SEQ ID NO:9),KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460),SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470),KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ IDNO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS(SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499),ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ IDNO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS(SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510),NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ IDNO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), HLAPSAV(SEQ ID NO:519), KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521),TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ IDNO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW(SEQ ID NO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532),DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ IDNO:535), and RYPLYWW (SEQ ID NO:536). The FGF-5-binding peptide in turnis chosen from CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC (PS2;SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538),CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ IDNO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP(SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546),TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ IDNO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP(SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557),GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ IDNO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQID NO:670) and TWIDSTP (SEQ ID NO:671). The TGFβ binding peptide in turnis chosen from CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF(MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC(SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565),QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ IDNO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568),acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570),NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ IDNO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ(SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581),ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ IDNO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC(SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673) andWTQHIHNC (SEQ ID NO:674). The TNFα binding peptide in turn is chosenfrom RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475), DMIMVSI(T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ ID NO:584),ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQ IDNO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV (SEQID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592), SQPIPI(SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).

In one embodiment, the invention provides for an isolated modifiedvariant Bowman Birk Protease Inhibitor (BBPI) that comprises asubstituted amino acid at least at one amino acid position chosen frompositions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50,52, 55, and 65 of the variant BBPI of SEQ ID NO:187, and in which thesecond protease inhibitory loop of the BBPI scaffold is a bindingpeptide. In some embodiments, the substituted amino acid at position 1chosen from 1A and 1C. In other embodiments, the substituted amino acidat position 4 is 4V. In other embodiments, the substituted amino acid atposition 5 is chosen from P and A. In other embodiments, the substitutedamino acid at position 13 is chosen from 13Y, 13I, 13F, 13M, 13L, 13V,13K, and 13R. In other embodiments, the substituted amino acid atposition 18 is chosen from 18I and 18V and 18L. In other embodiments,the substituted amino acid at position

25 is chosen from 25K, 25N, 25W, 25I, 25A and 25R. In other embodiments,the substituted amino acid at position 27 is chosen from 27H, 27K, 27V,27A, and 27Q. In other embodiments, the substituted amino acid atposition In other embodiments, the substituted amino acid at position 29is chosen from 29R, 29K, and 29P. In other embodiments, the substitutedamino acid at position 31 is chosen from 31Q, 31H, 31E, 31A, 31R, 31W,31K and 31T. In other embodiments, the substituted amino acid atposition 38 is chosen from 38N, 38K, and 38R. In other embodiments, thesubstituted amino acid at position 40 is chosen from 40H, 40K, 40Q, 40R,and 40Y. In other embodiments, the substituted amino acid at position 50is chosen from 50R, 50Q, 50K, 50T, 50V, 50M, and 50S. In otherembodiments, the substituted amino acid at position

In other embodiments, the substituted amino acid at position 52 ischosen from 52K, 52T, 52R, 52Q, 52L, 52H, 52A, 52M, 52S and 52E. Inother embodiments, the substituted amino acid at position 55 is 55M. Inother embodiments, the substituted amino acid at position 65 is chosenfrom 65E, 65Q, and 65D.

In other embodiments, the invention provides for an isolated modifiedvariant Bowman Birk Protease Inhibitor (BBPI) that comprises an insertof SEQ ID NO: 389, a substituted amino acid at least at one amino acidposition chosen from positions equivalent to 1, 4, 5, 11, 13, 18, 25,27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ IDNO:187, and in which the second protease inhibitory loop of the BBPIscaffold is a binding peptide.

In some embodiments, any one of the modified variant BBPIs describedabove is expressed as a fusion protein comprising the catalytic domainchosen from cellulase, cutinase and disulfide isomerase. In alternativeembodiments, the modified variant BBPI is expressed as the fusionprotein of SEQ ID NO:195.

In some embodiments, the modified variant BBPIs of the inventioncomprise a combination of at least two, at lest three, at least four, atleast five, at least six, at least seven, and at least eight amino acidsubstitutions chosen from positions equivalent to 1, 4, 5, 11, 13, 18,25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPI of SEQ IDNO:187. In some embodiments, the modified variant BBPI comprises acombination of at least two amino acid substitutions at amino acidpositions equivalent to positions 50 and 52 of SEQ ID NO:187. An exampleof a modified variant BBPI comprising a combination of two amino acidsubstitutions at positions 50 and 52 is the modified variant BBPI of SEQID NO:595. In other embodiments, the modified variant BBPI comprises acombination of at least three amino acid substitutions at amino acidpositions equivalent to positions 25, 29, 40, 50 and 52 of SEQ IDNO:187. In some embodiments, the combination of at least three aminoacids is chosen from combinations 25L-50T-52A, 29P-50T-52A, 40K-50T-52A.Examples of modified variant BBPIs comprising a combination of threeamino acid substitutions chosen from positions 25, 29, 40, 50 and 52 arethe modified variant BBPI of SEQ ID NOS: 603, 607 and 609. In otherembodiments, the modified variant BBPI comprises a combination of atleast four amino acid substitutions chosen from amino acid substitutionsat positions equivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187.In some embodiments, the combination of at least four amino acids ischosen from combinations 13I-25L-50T-52A, 13I-29P-50T-52A,13I-A0K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A.Examples of modified variant BBPIs comprising a combination of fouramino acid substitutions chosen from positions 13, 29, 50 and 52 are themodified variant BBPIs of SEQ ID NOS:596, 600, 602, 604, 606, 608, and643. In other embodiments, the modified variant BBPI comprises acombination of at least five amino acid substitutions chosen from aminoacid substitutions at positions equivalent to positions 13, 25, 29, 40,50, and 52 of SEQ ID NO:187. In some embodiments, the combination of atleast five amino acids is chosen from combinations 13I-25L-29P-50T-52A,13I-25L-40K-50T-52A, A13I-29P-40K-50T-52A, 25L-29P-40K-50T-52A,13I-29P-40K-50K-52A, and 13I-29P-40K-50T-52T. Examples of modifiedvariant BBPIs comprising a combination of five amino acid substitutionschosen from positions 13, 25, 29, 40, 50, and 52 are the modifiedvariant BBPIs of SEQ ID NOS: 432, 434, 443, 445, 446, 597, 599, 601,605, 615, 620, 624, and 625. In other embodiments, the modified variantBBPI comprises a combination of at least six amino acid substitutionschosen from amino acid substitutions at positions equivalent topositions 1, 4, 5, 11, 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. Insome embodiments, the combination of at least six amino acids is chosenfrom combinations 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A,4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A,13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A,13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A,13I-29P-38N-40K-50T-52A, and 13I-29P-40K-50T-52A-65E. Examples ofmodified variant BBPIs comprising a combination of six amino acidsubstitutions chosen from positions 1, 4, 5, 11, 13, 25, 29, 40, 50 and52 are the modified variant BBPIs of SEQ ID NOS: 598, 611, 612, 613,614, 616, 619, 621, 622, 623, and 626.

In other embodiments, the modified variant BBPI comprises a combinationof at least seven amino acid substitutions chosen from amino acidsubstitutions at positions equivalent to positions 13, 25, 29, 31, 40,50 and 52 of SEQ ID NO:187. In some embodiments, the combination of atleast seven amino acids is chosen from combinations13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A, and13I-27A-29P-31A-50K-52T. Examples of modified variant BBPIs comprising acombination of seven amino acid substitutions chosen from positions 13,25, 29, 31, 40, 50 and 52 are the modified variant BBPIs of SEQ IDNOS:491, 617, 618, and 632-639. In yet other embodiments, the modifiedvariant BBPI comprises a combination of eight amino acid substitutionschosen from amino acid substitutions at positions equivalent topositions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of eight amino acids is chosen fromcombinations 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K,13I-25K-27A-29R-31A-40H-50R-52L and 13I-25K-27Q-29P-31E-40H-50R-52Q.Examples of modified variant BBPIs comprising a combination of eightamino acid substitutions chosen from positions 13, 25, 27, 29, 31, 40,50 and 52 are the modified variant BBPIs of SEQ ID NOS: 627-631.

In some embodiments, the invention provides for an isolated modifiedvariant Bowman Birk Protease Inhibitor (BBPI) that binds VEGF(VEGF-BBPI). The VEGF-BBPI comprises a substituted amino acid at leastat one amino acid position chosen from positions equivalent to 1, 4, 5,11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variantBBPI of SEQ ID NO:187, and contains a VEGF-binding peptide that replacesthe second protease inhibitory loop. In some embodiments, theVEGF-binding peptide is chosen from SEQ ID NOS:9, 458, 459, 460, 468,469, 470, 471, 472 and 473. In some embodiments, the VEGF-BBPI has apolypeptide sequence chosen from SEQ ID NOS:601, 602, 627, 628, 630,631, 643, 491, 632, 633, 634, 635, and 636.

In some embodiments, the isolated modified variant BBPI of the inventionhas greater trypsin inhibitory activity than the correspondingunmodified precursor variant BBPI. In other embodiments, the isolatedmodified variant BBPI of the invention has greater trypsin inhibitoryactivity and production yield than the corresponding unmodifiedprecursor variant BBPI.

In some embodiments, the invention provides for an isolated modifiedvariant Bowman Birk Protease Inhibitor (BBPI) of SEQ ID NO:187 isfurther comprises a substituted amino acid at least at one amino acidposition chosen from positions equivalent to 13, 25, 27, 29, 31, 40, 50,and 52 of SEQ ID NO:187, and in which the VEGF-binding peptide isreplaced by a variant peptide to bind a target protein chosen fromFGF-5, TGFβ and TNFα.

In some embodiments, the VEGF-binding peptide comprised in the variantBBPI of SEQ ID NO:187 is replaced with an FGF-binding peptide having asequence chosen from SEQ ID NOS: 430, 431, 670, and 671. In otherembodiments, the VEGF-binding peptide is replaced with a TGFβ bindingpeptide chosen from SEQ ID NOS:436, 437, 438, 672, 673, and 674. In yetother embodiments, the VEGF-binding peptide is replaced with a TNFαbinding peptide chosen from SEQ ID NOS:474, 475, and 476.

The invention also encompasses modified variant BBPIs that compriseVEGF-, FGF5-, TGFβ- or TNFα-binding peptides that have greater trypsininhibitory activity and production yield than the correspondingunmodified precursor BBPI. In some embodiments, the modified variantBBPI comprising a FGF5-, TGFβ- or TNFα-binding peptide is chosen fromthe modified variant BBPIs of SEQ ID NOS:432, 434, 443, 445, 447, 637,638, and 639.

In another embodiment, the invention provides for an isolatedpolynucleotide that encodes a BBPI fusion protein. The polynucleotideencoding the BBPI fusion protein comprises a first polynucleotidesequence that encodes the catalytic unit of an enzyme, and a secondpolynucleotide sequence encoding a modified variant BBPI e.g. aVEGF-binding BBPI (VEGF-BBPI), an FGF-binding BBPI (FGF-BBPI), aTGF-binding BBPI (TGF-BBPI) or a TNF-binding BBPI (TNF-BBPI). In someembodiments, the first polynucleotide sequence encodes the catalyticunit of a cellulase.

In another embodiment, the invention provides for a method for amodified variant BBPI in a bacterial cell, which comprises the step ofmutating a polynucleotide that encodes a variant BBPI to generate apolynucleotide construct that encodes a modified variant BBPI comprisingan amino acid substitution at least at one amino acid position chosenfrom positions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38,40, 50, 52, 55, and 65 of the variant BBPI of SEQ ID NO:187; the step ofintroducing the polynucleotide construct into a bacterial host cell; thestep of culturing said bacterial cell under suitable culture conditionsto allow expression of said heterologous DNA sequence; and the step ofproducing the modified variant BBPI, which has a greater trypsininhibitory activity than the corresponding unmodified precursor variantBBPI. In some embodiments, the method of the invention further comprisesthe step of recovering the expressed modified variant BBPI. In otherembodiments, the method of the invention also comprises activating themodified variant BBPI.

In some embodiments, the modified variant BBPI has a greater trypsininhibitory activity and production yield than the correspondingunmodified precursor variant BBPI. Unmodified precursor variant BBPI arechosen from BBI-AV (SEQ ID NO:186), BBIt-AV (SEQ ID NO:187), BBdb-AV(SEQ ID NO:452), BBsb3 (SEQ ID NO:453), and BBtc-AV (SEQ ID NO:454), Insome embodiments, the second protease inhibitory loop of the modifiedvariant BBPI is a target protein binding peptide chosen from aVEGF-binding peptide, an FGF-5 binding peptide, a TGFβ binding peptideand a TNFα binding peptide.

Modified variant BBPIs that are produced by the method of the inventioninclude but are not limited to modified variant BBPIs that comprise acombination of amino acid substitutions that are chosen fromcombinations of three substitutions e.g. 25L-50T-52A, 29P-50T-52A,40K-50T-52A, four substitutions e.g. 13I-25L-50T-52A, 13I-29P-50T-52A,13I-A0K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A, 29P-40K-50T-52A, fivesubstitutions e.g. 13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A,25L-29P-40K-50T-52A,

13L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, and 13L-29P-40K-50T-52T, sixsubstitutions e.g. 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A,4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A,13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A,13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A,13I-29P-38N-40K-50T-52A, 13I-29P-40K-50T-52A-65E, seven substitutionse.g. 13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, and eight substitutions13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q,13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L,13I-25K-27Q-29P-31E-40H-50R-52Q.

In another embodiment, the invention provides for an expression vectorcomprising a polynucleotide sequence that encodes a modified variantBBPI of the invention. As described above, the modified variant BBPI isa Bowman Birk Protease Inhibitor in which the second protease inhibitoryloop has been replaced with a target protein binding peptide and whichis further modified to contain an amino acid substitution at least atone amino acid position chosen from positions equivalent to 1, 4, 5, 11,13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBPIof SEQ ID NO:187. In some embodiments, the second protease inhibitoryloop of the modified variant BBPI is a target protein binding peptidechosen from a VEGF-binding peptide, an FGF5-binding peptide, aTGFβ-binding peptide and a TNFα-binding peptide. In some embodiments,the modified BBPI is expressed as a protein having at least 80% identityto the polypeptide of SEQ ID NO:195.

In another embodiment, the invention provides for a host cell that istransformed with the vector of the invention, as described herein. Insome embodiments, the host cell is a bacterial cell e.g. a Bacillusspecies host cell.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D provides the DNA and amino acid sequences of theaprE-BCE103-BBI-Histag expression cassette (EcoRI-HindIII) cloned intothe pJM103 integration vector (SEQ ID NOS:1 and 2).

FIG. 2 provides a schematic map of the pJM103BBIhis expression vector.

FIG. 3 provides the DNA and amino acid sequences of 12BBIck81 from theBCE103 fusion site (at the BamHI) to the end of the gene (SEQ ID NOS:3and 4). The CK37281 peptide sequence (ACYNLYGWTC (SEQ ID NO:9) isinserted into both the trypsin and chymotrypsin inhibitory loops.

FIG. 4 provides a graph showing titers of active versus inactive2BBIck81 with various thiol reducing agents added at the givenconcentrations during the growth of the culture. The white portion ofthe bars represents the amount of active 2BBIck81 (determined by trypsininhibition) and the black portion of the bars represents the amount ofinactive 2BBIck81 (determined from BCE103 activity and assumes BCE103and 2BBIck 81 are produced in 1:1 molar ratio). The fraction of active2BBIck81 is indicated at the top of the bars. In this Figure, 37 C=noadditions, BME=2-mercaptoethanol, Cys=cysteine, Glut=reducedglutathione, DTT=dithiothreitol.

FIG. 5 provides a graph showing the activation of 2BBIck81 with2-mercaptoethanol (BME) after partial purification of theBCE-Ink2-2BBck81 fusion protein by ion exchange chromatography. Theconcentrations (μM) of BCE (black bars) and 2BBIck81 (white bars)measured by activity assays are shown before and after treatment with 3mM 2-mercaptoethanol (BME).

FIG. 6 provides a graph showing results from a competition analysis of2BBIck81 versus anti-VegF antibody for binding to VegF in a BioVerisassay. The binding competition with the CK37281 peptide is shown forcomparison and the binding with the wild-type BBI is included as anegative control.

FIG. 7A-D provides the sequence of the synthetic DNA fragment carryingthe H. insolens PDI-(hiPDI) that was inserted into the B. subtilis BBIexpression vector (using the BssHII and SacI sites), as well as theamino acid sequence (SEQ ID NOS:5 and 6)

FIG. 8A-D provides the DNA and amino acid sequences of the aprE-cutinaseexpression cassette that was ligated into the EcoRI-BamHI sites ofp2JM103-Ink2-2BBIck81 (SEQ ID NOS:7 and 8).

FIG. 9 provides the amino acid sequence of the unmodified BBIt-AVprotein (SEQ ID NO:187). Site-saturation libraries were constructed atall the numbered amino acids. The trypsin inhibitory loop and the VEGFbinding peptide loop (in italics) are indicated and disulfide bonds areshown by the connecting lines. At the bottom, the sequence of the VEGFbinding peptide is compared to the chymotrypsin inhibitory loop in sBBIthat it replaces. The downward arrow indicates the potential proteasecleavage site between the P₁ and P₁′ residues of the chymotrypsinreactive site.

FIG. 10 provides the relative BBI:BCE activity ratios for each aminoacid substitution made at position 29. The ratios were averaged fromquadruplicate cultures grown in microtiter plates in the absence (whitebars) or presence (black bars) of 2-mercaptoethanol. The activity ratiosfor sBBI are included for comparison as a positive control. The ratioswere normalized to the value determined for the wild-type amino acid inBBIt-AV (SEQ ID NO:187) (indicated by a horizontal line), leucine, thatis shown in the absence (horizontally lined bar) or in the presence(diagonally lined bar) of 2-mercaptoethanol.

FIGS. 11A-B respectively provide the relative BBI:BCE activity ratiosfor the amino acid substitutions made at position 50 (A) and position 37(B) (white bars). The ratios were averaged from quadruplicate culturesin the presence of 2-mercaptoethanol. The activity ratios for sBBI areincluded as a positive control for comparison (black bars). The ratiosare normalized to the value (indicated by a horizontal line) determinedfor the wild-type amino acids in BBIt-AV (SEQ ID NO:187) and is shownwith diagonally lined bars.

FIG. 12 provides the relative BBI:BCE activity ratios for the amino acidsubstitutions at the P₂′ position (residue 18 in FIG. 9, SEQ ID NO:187)in the trypsin inhibitory loop. The ratios were determined inquadruplicate in the presence of 2-mercaptoethanol (individual datapoints shown). The activity ratios for sBBI (BBI; SEQ ID NO:13), BBIt-AV(CT; SEQ ID NO187) and BBIt-AV-F50T (F50T) are shown on the right sideof the horizontal line and were added for comparison. The ratios arenormalized to the value (shown by a horizontal line) determined forBBIt-AV (CT).

FIG. 13 provides a comparison of the production of three BBIt-AVscontaining combinations of 8 amino acid substitutions: octuple variantBBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L (RL8; SEQ ID NO:630),BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q (QQ8; SEQ ID NO:628),and BBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T (KT8; SEQ IDNO:627). The amount of active BBI species was determined by trypsininhibition after growth (♦), after activation with 2-mercaptoethanol (▪)and after acid/heat treatment (▴). BBIt-AV, the quintuple variantBBIt-AV-A13I-L29P-A40K-F50T-V52A (TA5; SEQ ID NO:601) and sBBI areincluded for comparison.

FIG. 14 provides a comparison of the active BBI species (by trypsininhibition) after growth (black bars), after activation with2-mercaptoethanol (diagonally lined bars) and after acid/heat treatment(white bars) of different binding peptides (three TGFβ and two FGF5binders) in the precursor variant BBPI scaffold (designated parent (PT))versus the modified variant BBIt-AV-A13I-L29P-F50T-V52A scaffold (Q; SEQID NO:600). In all scaffolds, the binding peptides replaced thechymotrypsin inhibitory loop. PEN3 (SEQ ID NO:436), WTQ (SEQ ID NO:438)and MM021W (SEQ ID NO:437) are TGFβ binding peptides, and MM007 (SEQ IDNO:430) and FGFps2 (SEQ ID NO:431) are FGF5 binding peptides.

FIG. 15 provides the amino acid sequences of the variant Bowman BirkInhibitor scaffolds from Dolichos biflorus (BBdb-AV, SEQ ID NO:452), thevariant protease inhibitor IV or D-II (BBsb3-AV, SEQ ID NO:453) fromGlycine max (soybean), and from Torresea (Amburana) cearensis (BBtc-AV,SEQ ID NO:454) in which the chymotrypsin loop is replaced with aVEGF-binding peptide, that are aligned to the sequence of the variantBBI, BBIt-AV (SEQ ID NO:187). The differences from the BBIt-AV sequencebetween C9 and C58 are shown underlined in bold font. In all scaffolds,the CK3781 binding peptide (ACYNLYGWT; SEQ ID NO:9) replaces the secondprotease inhibitory loop.

FIG. 16 provides a comparison of the active BBI concentration (bytrypsin inhibition) of BBIt-AV, BBdb-AV and BBtc-AV after activationwith 2-mercaptoethanol (white bars) and after acid/heat treatment (blackbars).

FIG. 17 provides an alignment of unmodified variant BBIt-AV, (SEQ IDNO:187), BBdb-AV,(SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453) and BBtc-AV(SEQ ID NO:454) and the corresponding unmodified precursor BBIt (SEQ IDNO:187), BBsb3 (SEQ ID NO:449), BBtc (SEQ ID NO:450) and BBdb (SEQ IDNO:451) BBPIs. The star symbol (*) identifies the invariant conserved Cresidues. The numbered residues identify the amino acids that aresubstituted to generate the modified variant BBPIs of the invention.

FIG. 18 Identification of T-cell epitopes in wild type BBI. (A) Thestimulation indices (SI) values for all 71 donors were averaged and areshown, with standard deviations in error bars. (B) The percentresponders to each peptide in the 20 wt BBI peptide set are shown.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modified variant Bowman Birk ProteaseInhibitor proteins (BBPIs) that comprise peptides that bind targetproteins, and that are modified to have greater protease inhibitoryactivity and/or be produced at greater yields than the unmodified BBPIs.The invention encompasses polynucleotide constructs and expressionvectors containing polynucleotide sequences that encode the modifiedvariant BBPIs, the transformed host cells that express and produce themodified variant BBPIs, the modified variant BBPI proteins, thecompositions comprising the modified variant BBPIs, and the methods formaking and using the modified variant BBPIs in personal care.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, protein purification, protein engineering, protein and DNAsequencing, and recombinant DNA fields, which are within the skill ofthe art. Such techniques are known to those of skill in the art and aredescribed in numerous standard texts and reference works. All patents,patent applications, articles and publications mentioned herein arehereby expressly incorporated herein by reference in their entirety.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the present invention, some preferred methods andmaterials are described. Accordingly, the terms defined immediatelybelow are more fully described by reference to the Specification as awhole. It is to be understood that this invention is not limited to theparticular methodology, protocols, and reagents described, as these mayvary, depending upon the context they are used by those of skill in theart.

As used herein, the singular terms “a”, “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation and amino acid sequences are written left to right in aminoto carboxy orientation, respectively.

All patents, patent applications, and other publications, including allsequences disclosed within these references, referred to herein areexpressly incorporated by reference, to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference. Alldocuments cited are, in relevant part, incorporated herein by reference.However, the citation of any document is not to be construed as anadmission that it is prior art with respect to the present invention.

Numeric ranges are inclusive of the numbers defining the range. It isintended that every maximum numerical limitation given throughout thisspecification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to theSpecification as a whole. Accordingly, as indicated above, the termsdefined immediately below are more fully defined by reference to thespecification as a whole.

5.1 Definitions

As used herein, the terms “isolated” and “purified” refer to a nucleicacid or amino acid (or other component) that is removed from at leastone component with which it is naturally associated.

As used herein, the term “modified” when referring to a proteaseinhibitor (PI) e.g. Bowman Birk Protease Inhibitor (BBPI), refers to aBBPI having an amino acid sequence that is derived from the amino acidsequence of an unmodified “precursor” or “parent” BBPI protein. Theunmodified precursor BBPI can be a naturally-occurring or wild-typeprotein, or a variant BBPI. The amino acid sequence of the modifiedprotein is “derived” from the precursor protein amino acid sequence bythe substitution, deletion or insertion of one or more amino acids ofthe region of the precursor amino acid sequence. In some embodiments, atleast one amino acid is substituted to generate the modified proteaseinhibitor. In some embodiments, the parent protease inhibitor is avariant BBPI, which contains a trypsin and/or chotrypsin loop(s) thathas been replaced with a variant sequence. Substitution of at least oneamino acid of a variant precursor BBPI generates a modified variant BBPIprotease inhibitor. In some embodiments, the modified variant PIcomprises an amino acid substitution at least at one position equivalentto positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, and 65of SEQ ID NO:187(DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE: SEQID NO:187). In other embodiments, the modified variant PI comprises acombination of substitutions as described herein. In yet otherembodiments, the modified variant PI comprises an insertion. Suchmodifications are of the “precursor DNA sequence” which encodes theamino acid sequence of the precursor protease inhibitor rather thanmanipulation of the precursor protease inhibitor per se. The modifiedprotease inhibitors herein encompass the substitution of any of thenineteen naturally occurring amino acids at any one of the amino acidresidues in regions other than the reactive loops e.g. trypsin and/orchymotrypsin loops(s). The polynucleotides that encode the modifiedsequence are referred to as “modified polynucleotides”, and thepolynucleotides that encode the precursor protease inhibitor arereferred to as “precursor polynucleotides”.

As used herein, the term “protease inhibitor” (PI) herein refers to andis used interchangeably with Bowman Birk Protease Inhibitor (BBPI),which is a cysteine-rich protease inhibitor as described, for example,by Prakash et al. [J mol Evol 42:560-569 (1996)].

As used herein, the term “scaffold” refers to a BBPI protein sequenceinto which a variant sequence is introduced. In some embodiments, thescaffold is a variant scaffold, which has either the first proteaseinhibitory loop e.g. the trypsin loop, and/or the second proteaseinhibitory loop e.g. the chymotrypsin replaced with a binding peptidesequence. In other embodiments, the scaffold is a wild type BBPIscaffold The term “modified variant scaffold” refers to a variantscaffold that comprises modifications e.g. amino acid substitutions orinsertions in the backbone of the BBPI scaffold.

As used herein, the term “backbone” when used in reference to a variantBBPI scaffold refers to the portion of the variant BBPI that is outsideof the binding peptide sequence that has been introduced to replace thefirst and/or second protease inhibitory loop i.e. the trypsin and/orchymotrypsin loop. For example, the backbone of the variant BBPI of SEQID NO:187 refers to amino acids 1-40 and 50-66 i.e. the amino acidsN-terminal and C-terminal to the invariant cysteine residues at aminoacid positions 41 and 49, which bracket the VEGF binding sequence thatwas introduced to replace the chymotrypsin loop found in the wild typeBBPI i.e. BBI of SEQ ID NO:13(DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKEN;SEQ ID NO:13). In some embodiments, the scaffold has at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identity to the scaffold of the BBIt of SEQ ID NO:187.

As used herein, the terms “binding peptide”, “binding sequence”,“variant sequence” and “variant peptide” are used interchangeably, andrefer to the short polypeptide sequence(s) that replace the first and/orsecond protease inhibitory loops of protease inhibitor e.g. Bowman BirkInhibitor The binding peptide does not need to be of the same length asthe protease inhibitory loop sequence it is replacing in the scaffold,and it differs from the protease inhibitory loop sequence of thewild-type BBPI by at least two amino acids. In some embodiments,replacing the first and/or second protease inhibitory loop e.g. trypsinand/or chymotrypsin loops of a precursor protease inhibitor alters thesequence that is equivalent to that spanning amino acids 15 to 21(truypsin loop) and/or the sequence that is equivalent to that spanningamino acids 42 to 48 (chymotrypsin loop) of a wild-type e.g. SEQ IDNO:13 or variant SEQ ID NO:187 BBPI. The binding peptide sequence isheterologous to that of the protease inhibitor. Binding peptides bind totarget proteins.

A “VEGF binding peptide”, an “FGF binding peptide”, a “TGF bindingpeptide” and a “TNF binding peptide” herein refer to a peptide sequencethat binds VEGF, FGF5, TGFβ and TNFα, respectively.

A “VEGF composition”, an “FGF composition”, a “TGF composition” and a“TNF composition” herein refer to a composition e.g. a personal carecomposition, which comprises a VEGF-BBPI, an FGF-BBPI, a TGF-BBPI and aTGF-BBPI, respectively.

A “compound” when used in reference to a formulation described herein,refers to a modified variant BBPI e.g. a VEGF-BBPI, an FGF-BBPI, aTGF-BBPI or a TNF-BBPI. A formulation may include more than one type ofmodified variant BBPI i.e. the formulation may comprise a VEGF-BBPI anda TNF-BBPI, or any other combination of the four types of BBPIsdisclosed herein.

The term “a” when used in reference to a modified variant BBPI comprisedin a personal care composition, herein refers to a modified variant BBPIhaving a particular sequence. “a”, in this context does not limit thenumber of modified variant BBPI molecules that are needed in thepersonal care composition.

The terms “chymotrypsin loop” and “second protease inhibitory loop” areherein used interchangeably and refer to the sequence of amino acidsthat spans the amino acid sequence that corresponds to the secondreactive site loop of a protease inhibitor of the Bowman Birk family.For example, the second protease inhibitory loop of the wild-type BBPIinhibitor from Glycine max i.e. BBI of SEQ ID NO:13 is the peptidesequence that spans the second reactive site loop encompassed bycysteine 10 and cysteine 11 (see Prakash et al. supra). C10 and C11encompass an amino acid sequence that is equivalent to that which spansfrom amino acid 42 to amino acid 48 in the variant BBIt-AV of SEQ IDNO:187 (FIG. 9). The second protease inhibitory loop or chymotrypsinloop of a variant BBPI e.g. BBIt-AV of SEQ ID NO:187, corresponds to theamino acid sequence that spans amino acids 42-48 encompassed by thecysteines at positions 41 and 49, which are the cysteine residuesequivalent to C10 and C11 as described by Prakash et al. While the aminoacid sequence of second protease inhibitory loop of the wild-type BBI ofSEQ ID NO:13 is a chymotrypsin inhibitory peptide, the second proteaseinhibitory loop of the variant BBIt-AV of SEQ ID NO:187 is aVEGF-binding peptide. Thus, the terms “chymotrypsin loop” or “secondprotease inhibitory loop” herein refer to the position of the loop anddo not intend to imply that the sequence between C10 and C11 impartsprotease inhibitory activity to a variant BBPI. Similarly, the terms“trypsin loop” and “first protease inhibitory loop” are herein usedinterchangeably and refer to the sequence of amino acids that spans theamino acid sequence that corresponds to the first reactive site loop ofa protease inhibitor of the Bowman Birk family. For example, the secondprotease inhibitory loop of the wild-type BBPI inhibitor from Glycinemax i.e. BBI of SEQ ID NO:13 is the peptide sequence that spans thesecond reactive site loop encompassed by cysteine 4 and cysteine 5 (seePrakash et al. supra). C4 and C5 encompass an amino acid sequence thatis equivalent to that which spans from amino acid 15 to amino acid 21 inthe variant BBIt-AV of SEQ ID NO:187 (FIG. 9).

As used herein, the term “target protein” refers to protein (e.g.,enzyme, hormone, etc.), whose action would be blocked by the binding ofthe variant peptide. In some embodiments, the variant peptide binds thetarget protein when the peptide replaces the trypsin and/or chymotrypsinloop of a BBPI.

As used herein, “substituted” and “substitutions” refer toreplacement(s) of an amino acid residue or nucleic acid base in a parentsequence. In some embodiments, the substitution involves the replacementof a naturally occurring residue or base. In some embodiments, two ormore amino acids are substituted to generate a modified BBPI thatcomprises a combination of amino acid substitutions. In someembodiments, combinations of substitutions are denoted by the amino acidposition at which the substitution is made. For example, a combinationdenoted by 25-50-52 means that three amino acids at positions 25, 50 and52 are substituted. In other embodiments, the combination ofsubstitutions is denoted by the amino acid position and the amino acidresulting from the substitution. For example, a modified BBPI thatcomprises the combination of substitutions 13I-25L-50T-52A is a modifiedBBPI wherein the amino acid at position 13 has been substituted with anisoleucine, the amino acid at position 25 has been substituted with aleucine, the amino acid at position 50 has been substituted with athreonine, and the amino acid at position 52 has been substituted withan alanine. Amino acid positions are positions equivalent to thenumbered positions in the BBPI of SEQ ID NO:187. In some embodiments,the combination of substitutions is given in the context of the scaffoldin which the substitutions are made. For example, the modified variantBBPI of SEQ ID NO:601 is also referred to asBBIt-AV-13I-29P-40K-50T-52A, indicating that the BBIt-AV scaffold (SEQID NO:187) has been modified to contain the resulting substitutions atamino acids 13, 29, 40, 50, and 52. In some embodiments, the originaland substituted amino acid are indicated e.g. the modified variant BBPIof SEQ ID NO:601 can be referred to as BBIt-AV-A13I-L29P-A40K-F50T-V52A.

As used herein, “modification” and “modify” refer to any change(s) in anamino acid or nucleic acid sequence, including, but not limited todeletions, insertions, interruptions, and substitutions. In someembodiments, the modification involves the replacement of a naturallyoccurring residue or base. In other embodiments, the modificationcomprises a combination of at least one amino acid substitution. In yetother embodiments, the modification comprises an insertion with orwithout the insertion being combined with at least one amino acidsubstitution.

As used herein, the term “equivalent” when used in reference to an aminoacid residue or the position of an amino acid residue in a BBPI refersto the position of an amino acid residue in a modified BBPI thatcorresponds in position in the primary sequence of the unmodifiedprecursor BBPI. In order to establish the position of equivalent aminoacid positions in a BBPI, the amino acid sequence of the BBPI that ismodified is directly compared to the BBPI of SEQ ID NO:187, and inparticular to the cysteine residues that are known to be invariant inprotease inhibitors of the Bowman Birk Inhibitor family (Prakash et al.supra). After aligning the conserved cysteine residues, allowing forinsertions and deletions in order to maintain alignment (i.e. avoidingthe elimination of conserved cysteine residues through arbitrarydeletion or insertion), the residues at positions equivalent toparticular amino acid positions in the sequence of the BBPI of SEQ IDNO:187 are defined. Alignment of conserved cysteine residues preferablyshould conserve 100% of such residues. For example, in FIG. 17 the aminoacid sequences of variant and wild-type BBPIs are aligned to provide themaximum amount of homology between amino acid sequences. A comparison ofthese sequences shows that the invariant cysteine residues areconserved. While the primary sequence is the preferred structure fordetermining the position of equivalent amino acids in the BBPIs of theinvention, equivalent residues may also be identified by determininghomology at the level of tertiary structure for a protein BBPI protein.

Equivalent amino acid positions are defined as those for which theatomic coordinates of two or more of the main chain atoms of aparticular amino acid residue of the protein having putative equivalentresidues and the protein of interest (N on N, CA on CA, C on C and O onO) are within 0.13 nm and preferably 0.1 nm after alignment. Alignmentis achieved after the best model has been oriented and positioned togive the maximum overlap of atomic coordinates of non-hydrogen proteinatoms of the proteins analyzed. The preferred model is thecrystallographic model giving the lowest R factor for experimentaldiffraction data at the highest resolution available, determined usingmethods known to those skilled in the art of crystallography and proteincharacterization/analysis. The crystal structure of the Bowman Birkinhibitor from soybean has been determined (Hwang et al. J Biol. Chem.10; 252(3):1099-101 [1977], Wei et al., J Biol. Chem. 10; 254(11):4892-4[1979], Voss et al. Eur J Biochem. 15; 242(1):122-31 [1996]) and can beused as outlined above to determine equivalent amino acid positions onthe level of tertiary structure.

As used herein, “fusion polypeptides,” “fusion proteins,” and “fusionanalogs” encode from the amino-terminus a signal peptide functional as asecretory sequence functional in a host cell, a secreted polypeptide orportion thereof normally secreted from a host cell, a cleavable linkerpolypeptide and a desired polypeptide. In some embodiments, the fusionpolypeptides include a spacer peptide positioned between the secretorysequence and a secreted polypeptide. In some embodiments, the fusionprotein is processed by host cell enzymes (e.g., a protease), to yieldthe desired protein free from the other protein sequences in the fusionprotein. As used herein, the terms “fusion analog,” “fusionpolypeptide,” and “fusion protein” are used interchangeably.

As used herein, the term “activity” refers to any activity associatedwith a particular protein, such as enzymatic activity associated with aprotease. In some embodiments, the activity is biological activity. Infurther embodiments, activity encompasses binding of proteins toreceptors which results in measurable downstream effects (e.g., VEGFbinding to its cognate receptor). “Biological activity” refers to anyactivity that would normally be attributed to that protein by oneskilled in the art.

As used herein, “protease inhibitory activity” refers to the activity ofa BBPI in inhibiting the proteolytic activity of a protease i.e.inhibiting the ability of a protease to hydrolyze peptides or substrateshaving peptide linkages.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “production” with reference to a BBPI, encompasses the twoprocessing steps of a full-length protease including: 1. the removal ofthe signal peptide, which is known to occur during protein secretion;and 2. the removal of the pro region, which creates the active matureform of the BBPI and which is known to occur during the maturationprocess (Wang et al., Biochemistry 37:3165-3171 (1998); Power et al.,Proc Natl Acad Sci USA 83:3096-3100 (1986)).

As used herein, the term “production yield” refers to the level at whichan unmodified and/or modified variant protease inhibitor e.g. a variantBBPI is produced. The greater the production yield, the greater thelevel or amount of protease inhibitor that is produced.

As used herein, the term “efficient production” herein to the productionof a protein e.g. a modified variant BBPI, and implies that said proteinis produced at a level that is greater than that of an unmodified orprecursor variant BBPI.

As used herein, the term “substantially pure” when applied to theproteins or fragments thereof of the present invention means that theproteins are essentially free of other substances to an extent practicaland appropriate for their intended use. In particular, the proteins aresufficiently pure and are sufficiently free from other biologicalconstituents of the host cells so as to be useful in, for example,protein sequencing, and/or producing pharmaceutical preparations.

As used herein, the term “substantially free” encompasses preparationsof the desired polypeptide having less than about 20% (by dry weight)other proteins (i.e., contaminating protein), less than about 10% otherproteins, less than about 5% other proteins, or less than about 1% otherproteins.

As used herein, the terms “polynucleotide”, “nucleic acid molecule” and“nucleic acid sequence” include sequences of any form of nucleic acid,including, but not limited to RNA, DNA and cDNA molecules. It will beunderstood that, as a result of the degeneracy of the genetic code, amultitude of nucleotide sequences encoding a given protein may beproduced.

As used herein, the terms “DNA construct”, “polynucleotide construct”and “transforming DNA” are used interchangeably to refer to DNA used tointroduce sequences into a host cell or organism. The DNA may begenerated in vitro by PCR or any other suitable technique(s) known tothose in the art. In some embodiments, the DNA construct comprises asequence of interest (e.g., a modified sequence). In some embodiments,the sequence is operably linked to additional elements such as controlelements (e.g., promoters, etc.). In some embodiments, the DNA constructcomprises sequences homologous to the host cell chromosome. In otherembodiments, the DNA construct comprises non-homologous sequences. Oncethe DNA construct is assembled in vitro it may be used to mutagenize aregion of the host cell chromosome (i.e., replace an endogenous sequencewith a heterologous sequence).

As used herein, the term “heterologous DNA sequence” refers to a DNAsequence that does not naturally occur in a host cell. In someembodiments, a heterologous DNA sequence is a chimeric DNA sequence thatis comprised of parts of different genes, including regulatory elements.

As used herein, the term “heterologous protein” refers to a protein orpolypeptide that does not naturally occur in the host cell i.e. it isencoded by a heterologous sequence.

As used herein, “homologous protein” refers to a protein or polypeptidenative or naturally occurring in a cell.

As used herein, the term “vector” refers to a polynucleotide constructdesigned to introduce nucleic acids into one or more cell types. Vectorsinclude cloning vectors, expression vectors, shuttle vectors, andplasmids. In some embodiments, the polynucleotide construct comprises aDNA sequence encoding a modified variant BBPI.

As used herein, the term “expression vector” refers to a vector that hasthe ability to incorporate and express heterologous DNA fragments in aforeign cell. Many prokaryotic and eukaryotic expression vectors arecommercially available. Selection of appropriate expression vectors iswithin the knowledge of those of skill in the art.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in some eukaryotes orprokaryotes, or integrates into the host chromosome.

As used herein in the context of introducing a nucleic acid sequenceinto a cell, the term “introduced” refers to any method suitable fortransferring the nucleic acid sequence into the cell. Such methods forintroduction include but are not limited to protoplast fusion,transfection, transformation, conjugation, and transduction (See e.g.,Ferrari et al., “Genetics,” in Hardwood et al, (eds.), Bacillus, PlenumPublishing Corp., pages 57-72, [1989]).

As used herein, the terms “transformed” and “stably transformed” refersto a cell that has a non-native (heterologous) polynucleotide sequenceintegrated into its genome or as an episomal plasmid that is maintainedfor at least two generations.

As used herein, “percent (%) sequence identity” or “percent homology”when used in reference to a polynucleotide or to a polypeptide sequenceis defined as the percentage of nucleotide or amino acid residues in acandidate sequence that are identical with the nucleotide or amino acidresidues of a sequence disclosed herein. The percent identity shared bypolynucleotide or polypeptide sequences is determined by directcomparison of the sequence information between the molecules by aligningthe sequences and determining the identity by methods known in the art.In some embodiments, the alignment includes the introduction of gaps inthe sequences to be aligned. In addition, for sequences which containeither more or fewer nucleotides or amino acids than those of thecandidate polynucleotide or polypeptide sequences, it is understood thatthe percentage of homology will be determined based on the number ofhomologous nucleotides or amino acids in relation to the total number ofnucleotides or amino acids. Thus, for example, homology of sequencesshorter than those of the sequences identified herein will be determinedusing the number of nucleosites or amino acids in the shorter sequence.This homology is determined using standard techniques known in the art(See e.g., Smith and Waterman, Adv. Appl. Math., 2:482 [1981]; Needlemanand Wunsch, J. Mol. Biol., 48:443 [1970]; Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85:2444 [1988]; programs such as GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package (GeneticsComputer Group, Madison, Wis.); and Devereux et al., Nucl. Acid Res.,12:387-395 [1984]).

As used herein, an “analogous sequence” is one wherein the function ofthe protein is essentially the same as that designated for the BowmanBirk family of protease inhibitors. Additionally, analogous proteinsinclude at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% sequence identity with the sequence of the variant BBPI of SEQID NO:187. Analogous sequences are determined by known methods ofsequence alignment. A commonly used alignment method is BLAST, althoughas indicated above and below, there are other methods that also find usein aligning sequences.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng and Doolittle (Feng andDoolittle, J. Mol. Evol., 35:351-360 [1987]). The method is similar tothat described by Higgins and Sharp (Higgins and Sharp, CABIOS 5:151-153[1989]). Useful PILEUP parameters including a default gap weight of3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedby Altschul et al., (Altschul et al., J. Mol. Biol., 215:403-410,[1990]; and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787[1993]). A particularly useful BLAST program is the WU-BLAST-2 program(See, Altschul et al., Meth. Enzymol., 266:460-480 [1996]). WU-BLAST-2uses several search parameters, most of which are set to the defaultvalues. The adjustable parameters are set with the following values:overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched. However, the values may be adjusted toincrease sensitivity. A % amino acid sequence identity value isdetermined by the number of matching identical residues divided by thetotal number of residues of the “longer” sequence in the aligned region.The “longer” sequence is the one having the most actual residues in thealigned region (gaps introduced by WU-Blast-2 to maximize the alignmentscore are ignored). A preferred method utilizes the BLASTN module ofWU-BLAST-2 set to the default parameters, with overlap span and overlapfraction set to 1 and 0.125, respectively.

A “host cell” refers to a suitable cell from a cell that serves as ahost for an expression vector comprising DNA according to the presentinvention. A suitable host cell may be a naturally occurring orwild-type host cell, or it may be an altered host cell. In oneembodiment, the host cell is a Gram positive microorganism. In somepreferred embodiments, the term refers to cells in the genus Bacillus.

As used herein, “Bacillus sp.” includes all species within the genus“Bacillus,” as known to those of skill in the art, including but notlimited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

As used herein, a “promoter sequence” refers to a DNA sequence which isrecognized by the bacterial host for expression purposes. In preferredembodiments, it is operably linked to a DNA sequence encoding the fusionpolypeptide. Such linkage comprises positioning of the promoter withrespect to the translation initiation codon of the DNA sequence encodingthe fusion DNA sequence. In particularly preferred embodiments, thepromoter sequence contains transcription and translation controlsequences which mediate the expression of the fusion DNA sequence.

As used herein, a nucleic acid is “operably linked” when it is placedinto a functional relationship with another nucleic acid sequence. Forexample, DNA encoding a secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Operably linked DNAsequences are usually contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “personal care composition” refers to a productfor application to the skin, hair, nails, oral cavity and relatedmembranes for the purposes of improving, cleaning, beautifying,treating, and/or caring for these surfaces and membranes. In someembodiments, the personal care composition is in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersioning system, such as skin softener, a nutrient emulsion, anutrient cream, a massage cream, a treatment serum, a liposomal deliverysystem, a topical facial pack or mask, a surfactant-based cleansingsystem such as a shampoo or body wash, an aerosolized or sprayeddispersion or emulsion, a hair or skin conditioner, styling aid, or apigmented product such as makeup in liquid, cream, solid, anhydrous orpencil form. However, it is not intended that the present invention belimited to any particular form, as various forms find use in the presentinvention.

Personal care products can be classified/described as cosmetic,over-the-counter (“OTC”) compounds that find use in personal careapplications (e.g., cosmetics, skin care, oral care, hair care, nailcare). In some embodiments, the modified variant BBPI is added to apersonal care composition such as a hair care composition, a skin carecomposition, a nail care composition, a cosmetic composition, or anycombinations thereof.

As used herein, “skin care composition” refers to compositions that areapplied to skin in order to provide beneficial properties, including butnot limited to wrinkle minimizing, wrinkle removal, decoloring,coloring, skin softening, skin smoothing, depilation, cleansing, etc. Insome particularly preferred embodiments, the present invention providesskin care compositions that improve skin tone. In these embodiments, theimprovement comprises lessening of wrinkles, smoothing skin texture,modifying skin coloration, and other desired cosmetic benefits. Infurther embodiments, the skin care composition is in a form selectedfrom the group consisting of body washes, moisturizing body washes,deodorant body washes, antimicrobial cleansers, skin protectingtreatments, body lotions, facial creams, moisturizing creams, facialcleansing emulsions, surfactant-based facial cleansers, facialexfoliating gels, facial toners, exfoliating creams, facial masks, aftershave lotions, balms, and/or radioprotective compositions (e.g.,sunscreens).

As used herein, “cosmetic composition” refers to compositions that finduse in the cosmetics. The Food Drug and Cosmetic Act (FD&C Act)definition is used herein. Thus, cosmetics are defined by their intendeduse, as articles intended to be rubbed, poured, sprinkled, or sprayedon, introduced into, or otherwise applied to the human body forcleansing, beautifying, promoting attractiveness, or alteringappearance. These compositions provide non-therapeutic benefits and arenot regulated as pharmaceuticals. However, in some situations, cosmeticcompositions are incorporated into pharmaceutical compositions toprovide cosmetic benefits (e.g., products that treat skin or hairdiseases, but also contain cosmetic compositions for their coloring orother benefits). Cosmetic compositions include makeup compositions asdefined herein. Also, it is intended that the present inventionencompass the use of cosmetics on animals other than humans.

As used herein, the terms “pharmaceutical compositions” and “therapeuticcompositions” refer to compositions such as drugs that provide medicalbenefits, rather than solely cosmetic benefits. In the United States,pharmaceutical and therapeutic compositions are approved by the Food andDrug Administration for treatment and/or prevention of particularconditions.

As used herein, the term “drug” is defined as it is in the FD&C Actdefinition. Thus, drugs are defined as articles intended for use in thediagnosis, cure, mitigation, treatment or prevention of disease, andarticles (other than food) intended to affect the structure or anyfunction of the body of man or other animals.

As used herein, “leave-on” refers to a composition that is applied to asubject and not removed (e.g., cleansed by washing, rinsing, etc.) for aperiod of typically at least several hours (e.g., 4-12 hours) before thearea exposed to the composition is cleansed.

As used herein, a “rinse-off” composition is a composition that isapplied and cleansed (e.g., by washing, rinsing, etc.) soon after itsapplication (generally within about 30 minutes of application). In somepreferred embodiments, rinse-off compositions are formulated so as todeposit an effective amount of active(s) on the area treated.

As used herein, the term “cosmetic benefit” refers to a desired cosmeticchange that results from the administration of a personal carecomposition. Cosmetic benefits include but are not limited toimprovements in the condition of skin, hair, nails, and the oral cavity.In preferred embodiments, at least one cosmetic benefit is provided bythe skin care, hair care, nail care, and makeup compositions of thepresent invention.

As used herein, “cosmetically acceptable” refers to materials that aresuitable for use in contact with tissues of humans and/or other animals,without undue toxicity, incompatibility, instability, irritation,allergic responses, etc., commensurate with a reasonable benefit/riskratio.

As used herein, the terms “pigment,” “color pigment,” and “dye” used inreference to the compositions of the present invention encompasses anycompound that provides a color to the composition and/or imparts a colorto the surface (e.g., skin and/or hair) to which the composition isapplied.

The term “radioprotective” refers to a substance capable of blocking orfiltering. UV radiation sunscreens and sunblocks.

As used herein, “improving the appearance and/or condition of skin”refers to any benefit achieved through use of the personal carecompositions of the present invention. Examples of benefits include butare not limited to reducing the reducing imperfections and/or blemishesin skin color, including lightening hyperpigmented regions of skinand/or evening skin pigmentation, relieving dryness, eliminating rough,dry spots, improving the skin's ability to retain moisture and/orprotect itself from environmental stresses, reducing the appearance offine lines and wrinkles, improving appearance and skin tone, increasingskin firmness and/or suppleness, decreasing sagging of skin, increasingskin glow and clarity, increasing the skin renewal process, and/orremoving vellus hair. Improving the visual appearance of skin alsoencompasses regulating wrinkles, atrophy, skin lightening, skindarkening, skin smoothness, and/or reducing the visual appearance ofpores. In some embodiments, improving the appearance and/or condition ofthe skin results in skin improvements due to the treatment of a skindisorder with the personal care composition of the invention.

The term “angiogenesis” refers to the biological processes which resultin the development of blood vessels and/or increase in thevascularization of tissue in an organism.

The terms “angiogenic disease,” “angiogenic disorder,” and “angiogenicskin disorder,” are used in reference to a disorder, generally a skindisorder or related disorder which occurs as a consequence of or whichresults in increased vascularization in tissue. Oftentimes, the etiologyof the angiogenic disease is unknown. However, whether angiogenesis isan actual cause of a disease state or is simply a condition of thedisease state is unimportant, but the inhibition of angiogenesis intreating or reversing the disease state or condition is an importantaspect of the present invention. Thus, it is not intended that thepresent invention be limited to any particular mechanisms of action.Examples of angiogenic skin disorders which are suitable for treatmentutilizing compounds of the present invention include, but are notlimited to psoriasis, acne, rosacea, warts, eczema, hemangiomas andlymphangiogenesis, Sturge-Weber syndrome, neurofibromatosis, tuberoussclerosis, chronic inflammatory disease, and arthritis. Any skindisorder which has as a primary or secondary characterization, increasedvascularization, is considered an angiogenic skin disorder herein. Thus,the personal care compositions comprising VEGF-binding BBPIs(VEGF-BBPIs) provided by the present invention find use in treatment ofa wide variety of angiogenic skin disorders and/or conditions.

The term “rosacea” is used to describe acne, rosacea, or erythematosacharacterized by vascular and follicular dilation typically involvingthe nose and contiguous portions of the cheeks. Rosacea may vary fromvery mild but persistent erythema to extensive hyperplasia of thesebaceous glands with deep-seated papules and pustules and beaccompanied by telangiectasia at the affected erythematous sites. Thiscondition is also referred to as “hypertrophic rosacea” or “rhinophyma,”depending upon the severity of the condition. It is intended that theterm encompass all of the various forms of the condition.

The term “psoriasis” is used to describe a skin condition which ischaracterized by the eruption of circumscribed, discrete and confluent,reddish, silvery-scaled maculopapules. Although it is not intended thatthe present invention be limited to any particular body area, psoriaticlesions typically occur on the elbows, knees, scalp and trunk.Microscopically, these lesions demonstrate characteristic parakeratosisand elongation of rete ridges.

The term “acne” is used to describe a condition of the skincharacterized by inflammatory follicular, papular and pustular eruptionsinvolving the sebaceous apparatus. Although there are numerous forms ofacne, the most common form is known as acne simplex or acne vulgariswhich is characterized by eruptions of the face, upper back and chestand is primarily comprised of comedones, cysts, papules and pustules onan inflammatory base. The condition occurs primarily during puberty andadolescence due to an overactive sebaceous apparatus which is believedto be affected by hormonal activity.

The term “eczema” is a generic term used to describe acute or chronicinflammatory conditions of the skin, typically erythematous, edematous,papular, vesicular and/or crusting. These conditions are often followedby lichenification, scaling and occasionally, by duskiness of theerythema and, infrequently, hyperpigmentation. Eczema is oftenaccompanied by the sensation of itching and burning. Eczema vesiclesform due to intraepidermal spongiosis. Eczema is sometimes referred tocolloquially as “tetter,” “dry tetter,” and “scaly tetter.” There arenumerous subcategories of eczema, all of which are treated by one ormore of the compounds according to the present invention.

The term “hemangioma” refers to a benign self-involuting tumour ofendothelial cells (the cells that line blood vessels). Hemangiomas areconnected to the circulatory system and filled with blood. Theappearance depends on location. If they are on the surface of the skinthey look like a ripe strawberry, if they are just under the skin theypresent as a bluish swelling. Sometimes they grow in internal organssuch as the liver or larynx. Approximately 80% are located on the faceand neck, with the next most prevalent location being the liver. Thepersonal care compositions of the invention are intended to treathemangiomas of the skin i.e. hemangiomas that are on the surface of theskin and hemangiomas that are just under the skin.

The term “scleroderma” herein refers to a chronic disease characterizedby excessive deposits of collagen in the skin or other organs.Scleroderma affects the skin, and in more serious cases it can affectthe blood vessels and internal organs. The most evident symptom isusually the hardening of the skin and associated scarring. The skin mayappear tight, reddish or scaly. Blood vessels may also be more visible.A significant player in the process is transforming growth factor(TGFβ). Topical treatment for the skin changes of scleroderma do notalter the disease course, but may improve pain and ulceration.

As used herein, “hair care composition” refers to compositions that areapplied to hair to provide beneficial properties such as thickening,thinning, coloring, decoloring, cleansing, conditioning, softening,shaping, etc. In some embodiments, the hair care composition is in aform selected from the group consisting of shampoos, conditioners,anti-dandruff treatments, styling aids, styling conditioners, hairrepair or treatment sera, lotions, creams, pomades, and chemicaltreatments. In other embodiments, the styling aids are selected from thegroup consisting of sprays, mousses, rinses, gels, foams, andcombinations thereof. In further embodiments, the chemical treatmentsare selected from the group consisting of permanent waves, relaxers, andpermanents, semi-permanents, temporary color treatments and combinationsthereof.

As used herein, “inhibiting hair growth” and “inhibition of hair growth”refer to an observed lessening of hair length and/or thickness. Thus, insome preferred embodiments, application of a personal care compositionof the present invention provides a benefit in lessening hair lengthand/or thickness as compared to an area in which a personal carecomposition of the present invention has not been applied. In someembodiments, the observed reduction of hair growth and/or thickness is arange from less than 1% to more than 99%, as compared to untreatedareas, while in other embodiments, the observed reduction is from about100% to about 90%, from about 90% to about 80%, from about 80% to about70%, from about 70% to about 60%, from about 60% to about 50%, fromabout 50% to about 40%, from about 40% to about 30%, from about 30% toabout 20%, from about 20% to about 10%, from about 10% to about 1%.Indeed, it is not intended that the term be limited to any particularpercentage reduction, as long as the reduction is observable by visual(i.e., by eye) or other means. It is also intended that the termencompass “preventing hair growth” to any degree, as described above. Itis not intended that the term be limited to the complete prevention ofhair growth (i.e., there is no observed growth of hair).

The terms “dermatological inflammatory disorder” or “inflammatory skindisorder” refer to skin condition associated with inflammation of theskin. In some embodiments, the inflammatory skin disorder is a disorderassociated with elevated levels of inflammatory cytokines e.g. TNFα.

As used herein, in some embodiments, the term “compound” refers to theBBPI comprised in the personal care compositions of the invention.

The term “effective amount” is used throughout the specification todescribe concentrations or amounts of compounds according to the presentinvention which may be used to produce a favorable change in the diseaseor condition treated, e.g. whether that change is hair growth,prevention of hair growth or for ameliorating a condition caused orassociated with a disorder. As used herein, “safe and effective amount”refers to a sufficient amount of a material that significantly induces apositive modification to the area upon which the material is applied andalso does not result in the production of serious side effects (at areasonable risk/benefit ratio). The safe and effective amount of thematerial may vary with the particular skin or other body part beingtreated, the age of the subject being treated, the severity of thecondition being treated, the duration of treatment, the nature ofconcurrent therapy, the specific material used, the particular carrierutilized, etc. Those of skill in the art are capable of adjusting theconcentration of the personal care compositions provided herein for thedesired application of the compositions.

As used herein, “active” (and “actives”) refers to a composition thatimparts a benefit to a subject being treated. For example, in someembodiments, the present invention provides personal care compositionscomprising a modified variant BBPI, e.g. modified variant VEGF-BBPI, a“primary active” which functions to provide benefit to the area to whichit is applied. Thus, in some embodiments, the modified variant VEGF-BBPIis present in skin care formulations and serves to treat the skin ofsubjects suffering from an angiogenic skin disorder. It is not intendedthat the term be limited to VEGF-BBPI, as there are additionalconstituents present in the personal care compositions of the presentinvention which impart benefits. In some embodiments, these additionalconstituents are encompassed by the designation “secondary actives.”Primary and secondary actives are collectively referred to as “actives”herein. Other “primary actives” provided by the invention includeFGF-BBPIs, TGF-BBPIs and TNF-BBPIs.

As used herein, “vitamin B₃ compound” means a compound having theformula:

wherein R is —CONH₂ (i.e., niacinamide), —COOH (i.e., nicotinic acid) or—CH₂OH (i.e., nicotinyl alcohol); derivatives thereof; and salts of anyof the foregoing.

As used herein, “non-vasodilating” means that an ester does not commonlyyield a visible flushing response after application to the skin in thesubject compositions. It is contemplated that the majority of thegeneral population would not experience a visible flushing response,although such compounds may cause vasodilation not visible to the nakedeye.

As used herein, “retinoid” includes all natural and/or synthetic analogsof Vitamin A and/or retinol-like compounds which possess the biologicalactivity of Vitamin A in/on the skin, as well as the geometric isomersand stereoisomers of these compounds. However, it is not intended thatthe term be limited to these compounds, as the term encompasses vitaminA alcohol (retinol) and its derivatives such as vitamin A aldehyde(retinal), vitamin A acid (retinoic acid) and vitamin A esters (e.g.,retinyl acetate, retinyl propionate and retinyl palmitate), etc. It isfurther intended that the term encompass all-trans-retinoic acids and13-cis-retinoic acids. It is also intended that the term encompasscompositions that are encapsulated, as well as provided for use invarious forms. The terms “retinol” and “retinal” preferably comprise theall-trans compounds. The retinoid preferably used for the formulation ofthe present invention is all-trans-retinol, generally referred to as“retinol” herein.

As used herein, “carotenoid” is used in reference to β-carotene,lycopene, lutein, astaxanthin, zeaxanthin, cryptoxanthin,citranaxanthin, canthaxanthin, bixin, β-apo-4-carotenal,β-apo-8-carotenal, β-apo-8-carotenoic esters, alone, as well as incombination. Carotenoids which are preferably used are β-carotene,lycopene, lutein, astaxanthin, zeaxanthin, citranaxanthin andcanthaxanthin. In some embodiments, carotenoids are utilized incrystalline form, as well as in formulations, including but not limitedto dry powders (See e.g., dry powders, as described in EP 0 065 193;hereby incorporated by reference). In some embodiments, the preferreduse in the case of lycopene, astaxanthin and canthaxanthin is oflycopene-, astaxanthin- and canthaxanthin-containing dry powders, forexample LYCOVIT®, LUCANTIN® Pink and LUCANTIN® Red (10% dry powdersrespectively of lycopene, astaxanthin and canthaxanthin, commerciallyavailable from BASF AG, Ludwigshafen, Germany. As used herein, the term“dispersed phase” is used as by those of skill in the art of emulsiontechnology as the phase that exists as small particles or dropletssuspended in and surrounded by a continuous phase. The dispersed phaseis also known as the “internal” or “discontinuous” phase.

As used herein, “penetration enhancers” refer to compositions thatfacilitate penetration through the upper stratum corneum barrier to thedeeper skin layers. Examples of penetration enhancers include, but arenot limited to, propylene glycol, azone, ethoxydiglycol, dimethylisosorbide, urea, ethanol, dimethyl sulfoxide, microemulsions,liposomes, and nanoemulsions.

As used herein, the terms “emulsifier” and “surfactant” refer tocompounds that disperse and suspend the dispersed phase within thecontinuous phase of a material. Surfactants find particular use inproducts intended for skin and/or hair cleansing. In particularembodiments, the term “surfactant(s)” is used in reference tosurface-active agents, whether used as emulsifiers or for othersurfactant purposes such as skin cleansing.

In various embodiments, the present invention also includes“protectants” such as UV absorbers (e.g., octyl methoxycinnamate,benzophenone-3, titanium dioxide, and octyl salicylate); film-formingagents (e.g., VP/Eicosene copolymer); cosmeceutical agents (e.g.,peptides and proteins, alpha hydroxy acids, and retinol and retinoicacid derivatives); antioxidants (e.g., tocopherol and derivativesthereof and ascorbic acid and derivatives thereof); vitamins (e.g., B,D, K and their derivatives); antiperspirant actives (e.g., aluminumhydroxide and zirconium hydroxide); depilating agents (e.g.,thioglycolate salts); anti-acne agents (e.g., salicylic acid and benzoylperoxide); abrasives and exfoliants (e.g., silicates, pumice, andpolyethylene); and extracts of plant, fruit, vegetable and/or marinesources.

As used herein, the term “bioactivity” refers to a cause and effectrelationship between a composition and a biological system. Thus, theterm is used as by those skilled in the art of biotechnology andbiological sciences as the phrase that describes a cause and effectrelationship between a molecular composition and living biologicalmatter (e.g., tissue, cells, etc.).

As used herein as a noun, the term “bioactive” refers a composition thatexhibits bioactivity upon administration to living biological matter(e.g., tissue, cells, etc.). The term is used synonymously with“bioactive compound.”

As used herein, “silicone gum” means high molecular weight siliconeshaving an average molecular weight in excess of about 200,000 andpreferably from about 200,000 to about 4,000,000. It is intended thatthe definition encompass non-volatile polyalkyl and polyaryl siloxanegums.

As used herein, a “composition comprising a modified variant BBPI”refers broadly to any composition containing the given modified variantBBPI. The composition may be in any form, particularly a form that issuitable for administration.

As used herein, a compound is said to be “in a form suitable foradministration” when the compound may be administered to a human orother animal by any desired route (e.g., topical, oral, etc.).

As used herein, “safe and effective amount” refers to a sufficientamount of a material that significantly induces a positive modificationto the area upon which the material is applied and also does not resultin the production of serious side effects (at a reasonable risk/benefitratio). The safe and effective amount of the material may vary with theparticular skin or other body part being treated, the age of the subjectbeing treated, the severity of the condition being treated, the durationof treatment, the nature of concurrent therapy, the specific materialused, the particular carrier utilized, etc. Those of skill in the artare capable of adjusting the concentration of the personal carecompositions provided herein for the desired application of thecompositions.

5.2 Bowman Birk Protease Inhibitor Scaffolds

Bowman Birk Protease Inhibitor proteins (BBPI) are a kinetically andstructurally well-characterized family of small proteins (60-90residues), and have been found only in the seed of monocotyledous anddicotyledonous plants, and have not been identified in any other part ofthe plant (See e.g., Birk, Int. J. Pept. Protein Res., 25:113-131[1985]). The sequences of many wild-type BBPI scaffolds have beendetermined from both monocotyledonous and dicotyledonous seeds, and havebeen analyzed (Prakash et al., J mol Evol 42:560-569 [1996]). Theytypically have a symmetrical structure of two tricyclic domains eachcontaining an independent binding loop, although some have one domainand some have more than two domains. The major ˜8 kDa Bowman BirkInhibitor isolated from soybeans (BBI) has two separate reactive siteloops, loop I inhibits proteases having trypsin-like specificity andloop 11 inhibits proteases with chymotrypsin-like specificity (See e.g.,Chen et al., J. Biol. Chem., 267:1990-1994 [1992]; Werner and Wemmer,Biochem., 31:999-1010 [1992]; Lin et al., Eur. J. Biochem., 212:549-555[1993]; Voss et al., Eur. J. Biochem., 242:122-131 [1996]; and Billingset al., Pro. Natl. Acad. Sci., 89:3120-3124 [1992]). These bindingregions each contain a “canonical loop” structure, which is a motiffound in a variety of serine proteinase inhibitors (Bode and Huber, Eur.J. Biochem., 204:433-451 [1992]). In some embodiments, wild-type BBPIscaffolds serve as wild-type precursor BBPI scaffolds from which variantand modified variant BBPI scaffolds are derived.

In some embodiments, the present invention provides for variant BBPIscaffolds in which the trypsin (loop I) and/or chymotrypsin loop(s)(loop II) is replaced by a variant peptide. In some embodiments, thetrypsin loop is replaced with a variant peptide resulting in a variantBBPI that retains chymotrypsin inhibitory activity (CIA). In otherembodiments, the chymotrypsin loop is replaced with a variant peptide togenerate a variant BBPI that retains trypsin inhibitory activity (TIA).In yet other embodiments, both the trypsin and the chymotrypsin loops ofthe BBPI are each replaced with a variant peptide. Non-limiting examplesof BBPI scaffolds in which the trypsin and/or chymotrypsin loop isreplaced with a variant peptide sequence include wild-type andunmodified variant scaffolds. Examples of wild-type precursor scaffoldsinclude but are not limited to the scaffolds disclosed by Prakash(supra), such as the scaffold of the soybean inhibitor from Glycine max(BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ IDNO:185;DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSE), theinhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449;PSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCVCTFSIPAQCVCVDMKDFCYEPCK; thesoybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450;DDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCMCTRSQPGQCRCLDTNDFCYKPCKSRDD) andthe inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451;SSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACTHSIPAQCRCFDITDFCYKPCSG).Examples of unmodified variant precursor scaffolds include but are notlimited to scaffolds in which the chymotrypsin loop has been replacedwith a VEGF binding variant sequence include the BBI-AV (SEQ ID NO:186;DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSEDDKEN),BBIt-AV (SEQ ID NO:187;DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE),BBdb-AV (SEQ ID NO:452;DPSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCACYNLYGWTCVCVDMKDFCYEPCK),BBsb3-AV (SEQ ID NO:453;DPDDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCACYNLYGWTCRCLDTNDFCYKPCKSRDD),and BBtc-AV (SEQ ID NO:454;DPSSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACYNLYGWTCRCFDITDFCYKPCSG),BBIt-VEGK (SEQ ID NO:640;DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYYLYWWCKCTDITDFCYEPCKPSE),BBIt-VEGT (SEQ ID NO:641;DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACTLWKSYWCKCTDITDFCYEPCKPSE)and BBIt-VEGKD (SE ID NO:642;DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYDLYWWCKCTDITDFCYEPCKPSE).In some embodiments, unmodified variant precursor scaffolds are variantscaffolds in which a variant peptide replaces the chymotrypsin loop ofthe wild-type BBPI and which also introduces a substitution of the aminoacid at the position equivalent to position 40 of the BBPI of SEQ IDNO:187. For example, the unmodified variant BBPI scaffold of SEQ IDNO:187 (BBIt-AV) was derived from the wild-type precursor scaffold ofSEQ ID NO:185 by replacing the chymotrypsin loop of SEQ ID NO:185 withthe VEGF variant peptide of SEQ ID NO:9, which introduces an amino acidsubstitution I40A in addition to replacing the chymotrypsin loop. Theunmodified variant BBPI of SEQ ID NO:187 is modified to generate amodified variant BBPI, which in addition to the replaced chymotrypsinloop, comprises at least one amino acid substitution as described below.

Although numerous isoforms of BBI have been characterized, SEQ ID NO:13is an example of the amino acid sequence of the wild-type BBI scaffoldused in some embodiments comprising approximately 71 amino acid residues(See Example 1). In some embodiments, the invention provides for BBPIscaffolds e.g. SEQ ID NO:11, that include the pro region, while in otherembodiments, the invention provides for BBI scaffolds from which the propeptide has been removed e.g. SEQ ID NO:185. In yet other embodiments,the invention provides for BBI scaffolds from which up to 10 amino acidshave been removed from the N- or C-terminus. In some embodiments, theinvention provides for BBI scaffolds from which up to 5 e.g. SEQ IDNO:187. It will be appreciated that truncations of the BBI scaffold willnot destroy the ability of the BBI to bind the target protein.

In soybeans, BBPIs e.g. BBI is produced as a pro-protein with anN-terminal pro-peptide that is 19 amino acids in length. Thus, in someembodiments, BBI is produced with all or at least a portion of thepropeptide. In some embodiments, BBI is truncated, with as many as 10amino acid residues being removed from either the N- or C-terminal. Forexample, upon seed desiccation, some BBPI molecules have the C-terminal9 or 10 amino acid residues removed. Thus, proteolysis is generallyhighly tolerated prior to the initial disulfide and just after theterminal disulfide bond, the consequences of which are usually notdetrimental to the binding to target protein. However, it will beappreciated that any one of the isoforms or truncated forms a BBPI finduse in various embodiments of the present invention. In someembodiments, the truncated form of BBPI that finds use in the presentinvention is the variant BBIt in which the chymotrypsin loop is replacedwith a variant sequence as described below.

5.3 Variant BBPIS

As indicated above, BBPIs have binding loops (i.e. trypsin andchymotrypsin loops) that inhibit proteases. The present inventionprovides variant BBPIs, which are derived from wild-type or fromunmodified variant BBPI precursor scaffolds in which one or morereactive sites (e.g., Loop I (trypsin) and/or Loop II (chymotrypsin) ofBBPIs have been replaced with variant peptides that bind a targetprotein. Non-limiting examples of target proteins that are bound byvariant peptides comprised in the BBPIs of the invention include variouscytokines, including cytokines of the tumor necrosis factor (TNF)family, particularly TNF-α; cytokines of the transforming growth factorfamily, particularly TGFβ; cytokines of the fibroblast growth factorfamily (FGF), particularly FGF-5, and cytokines of the vascularendothelial growth factor (VEGF) family, particularly VEGF-A. Inaddition, variant peptides that replace one or both loops of the BBPIsof the invention include peptides that interact with inhibitors of thecomplement pathway such as C2, C3, C4 or C5 inhibitors, Compstatin, andother proteins of interest. Indeed, it is not intended that the presentinvention be limited to any particular sequence substituted into eitherof these loops, as any suitable sequence finds use in the presentinvention.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPIprecursor scaffold is replaced with variant sequence that binds VEGF togenerate variant VEGF-BBPI proteins. In some embodiments, the variantBBPI is derived from a wild-type or an unmodified variant BBPI precursorscaffold chosen from the scaffolds of the soybean inhibitor from Glycinemax (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ IDNO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), thesoybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), theinhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), theBBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ IDNO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffoldof (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), theBBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ IDNO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, anywild-type BBPI precursor scaffolds, such as those disclosed by Prakashet al. (supra), may be used to generate variant BBPI scaffolds.

In some embodiments, the VEGF variant sequences include, but are notlimited to VEGF-binding peptides disclosed in U.S. application Ser. Nos.09/832,723 and 10/984,270, including peptides ACYNLYGWTC (SEQ ID NO:9),KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460),SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470),KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ IDNO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS(SEQ ID NO:497), ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499),ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ IDNO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS(SEQ ID NO:508), NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510),NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ IDNO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), andHLAPSAV (SEQ ID NO:519). In some other embodiments, the VEGF variantsequences include, but are not limited to VEGF-binding peptidesdisclosed in U.S. application Ser. No. 11/919,717, including peptidesKYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ IDNO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW(SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ ID NO:530),KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ IDNO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW(SEQ ID NO:536).

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPIprecursor scaffold is replaced with variant sequences that interact withFGF5 to generate variant FGF-BBPI proteins. In some embodiments, thevariant BBPI is derived from a wild-type or an unmodified variant BBPIprecursor scaffold chosen from the scaffolds of the soybean inhibitorfrom Glycine max (BBI; SEQ ID NO:13) or the mature and truncated formthereof (SEQ ID NO:185), the inhibitor from Dolichos biflorus (BBdb; SEQID NO:449), the soybean inhibitor D-II from Glycine max (BBsb3; SEQ IDNO:450), the inhibitor from Torresea (Amburana) cearensis (BBtc; SEQ IDNO:451), the BBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of(SEQ ID NO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AVscaffold of (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454),the BBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of(SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). Inaddition, any wild-type BBPI precursor scaffolds, such as thosedisclosed by Prakash et al. (supra), may be used to generate variantBBPI scaffolds.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPIprecursor scaffold is replaced with variant sequences that interact withFGF5. In some embodiments, the FGF5 variant sequences include, but arenot limited to FGF5-binding peptides disclosed in U.S. application Ser.Nos. 10/984,410 and 12/033,848, including peptides CACRTQPYPLCF (MM007;SEQ ID NO:430), CICTWIDSTPC(PS2; SEQ ID NO:431), CYGLPFTRC (SEQ IDNO:537), CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC(SEQ ID NO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542),CRIHLAHKC (SEQ ID NO:543), TNIDSTP(SEQ ID NO:544), HLQTTET (SEQ IDNO:545), SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQID NO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN(SEQ ID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553),TNIDSTP (SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ IDNO:556), PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQID NO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP(SEQ ID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671).

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPIprecursor scaffold is replaced with variant sequences that interact withTGFβ to generate variant TGF-BBPIs. In some embodiments, the variantBBPI is derived from a wild-type or an unmodified variant BBPI precursorscaffold chosen from the scaffolds of the soybean inhibitor from Glycinemax (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ IDNO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), thesoybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), theinhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), theBBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ IDNO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffoldof (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), theBBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ IDNO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, anywild-type BBPI precursor scaffolds, such as those disclosed by Prakashet al. (supra), may be used to generate variant BBPI scaffolds.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPIprecursor scaffold is replaced with variant sequences that interact withTGFβ. In some embodiments, the TGFβ variant sequences include, but arenot limited to TGFβ-binding peptides disclosed in U.S. application Ser.No. 10/581,142, including peptides CLCPENINVLPCN (PEN3; SEQ ID NO:436),CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438),CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ IDNO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD(SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568),acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570),NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ IDNO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ(SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581),ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ IDNO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC(SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673) andWTQHIHNC (SEQ ID NO:674).

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPIprecursor scaffold is replaced with variant sequences that interact withTNFα to generate variant TNF-BBPIs. In some embodiments, the variantBBPI is derived from a wild-type or an unmodified variant BBPI precursorscaffold chosen from the scaffolds of the soybean inhibitor from Glycinemax (BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ IDNO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), thesoybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), theinhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), theBBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ IDNO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffoldof (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), theBBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ IDNO:641) and the BBIt-VEGKD scaffold of (SE ID NO:642). In addition, anywild-type BBPI precursor scaffolds, such as those disclosed by Prakashet al. (supra), may be used to generate variant BBPI scaffolds.

In some embodiments, the trypsin and/or chymotrypsin loop(s) of the BBPIprecursor scaffold is replaced with variant sequences that bind TNFα. Insome embodiments, the TNFα binding sequences include, but are notlimited to TNF-binding peptides disclosed in U.S. application Ser. No.10/968,732, including peptides RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2;SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583),ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ IDNO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH(SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).

In some embodiments, the variant BBPIs further comprises a peptideinsert that is positioned at the N-terminus of the modified variantBBPI. In some embodiments, the peptide insert comprises a sequence ofbetween 1 and 15 amino acids. In other embodiments, the peptide insertcomprises a sequence between 5 and 10 amino acids. In some embodiments,the peptide insert comprises the peptide of SEQ ID NO:389(DDEPSKPCCDPDP; SEQ ID NO:389). Examples of modified variant BBPIs thatcomprise the peptide insert of SEQ ID NO:389 are the modified variant4D13BBIt-AV of (DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE; SEQ ID NO:390), and the modified variantBBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413(DPDDEPSKPCCDPDPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE; SEQ ID NO:413).

In some embodiments, variant sequences are selected by various methodsknown in the art, including but not limited to phage display and othersuitable screening methods. For example, a random peptide gene libraryis fused with phage PIII gene so the peptide library will be displayedon the surface of the phage. Subsequently, the phage display library isexposed to the target protein and washed with buffer to removenon-specific binding (this process is sometimes referred to as panning).Finally, the binding phage and PCR the DNA sequence for the peptideencoded are isolated.

In most embodiments, one of the loops is replaced with a variantsequence i.e., peptides often 3 to 14 amino acids in length, with 5 to10 amino acids being preferred, to generate the variant BBPI. Longersequences find use in the present invention, as long as they provide thebinding and/or inhibition desired. In addition, peptides suitable foruse as replacements of the binding loop(s) preferably adopt a functionalconformation when contained within a constrained loop (i.e., a loopformed by the presence of a disulfide bond between two cysteineresidues). In some specific embodiments, the peptides are between 7 and9 amino acids in length. In other embodiments, the variant sequences arepeptides of 10 amino acids in length.

5.4 Modified Variant BBPI Proteins

5.4.1 Modified Variant BBPIs: Single Amino Acid Substitutions

In some embodiments, the invention provides for modified variant BBPIs,which are variant BBPIs that further comprise at least one amino acidsubstitution in the backbone of the BBPI. Thus, in some embodiments,modified variant BBPIs are variant BBPIs that contain trypsin and/orchymotrypsin loop(s) that have been replaced by a variant sequence thatbinds to a target protein, and that are further altered by comprising atleast one amino acid substitution C-terminal and/or N-terminal to thereplaced loop. Thus, in some embodiments, modified variant BBPIs are thevariant BBPIs described in section 5.4 in which the trypsin and/orchymotrypsin loop(s) has been replaced by a variant peptide, but thatfurther comprise a substituted amino acid at least at one positionequivalent to a position chosen from positions equivalent to positions1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQID NO:187, as described in section 5.4.

An amino acid residue of a modified variant BBPI is at an equivalent tothe position of a residue of a precursor BBPI if it is homologous (i.e.corresponding in position in primary structure) to a specific residue.In order to establish homology to primary structure, the amino acidsequence of a precursor BBPI is directly compared to the primary aminoacid sequence, and particularly to the set of cysteine residues known tobe conserved in BBPIs for which the sequence is known. FIG. 17 shows theconserved cysteine residues among exemplary wild type and variantprecursor BBPIs described herein. Equivalent residues that aresubstituted in the modified variant BBPIs of the invention are numbered.

The precursor BBPI may be a naturally-occurring BBPI or a variant BBPI.Specifically, such modified variant BBPIs have an amino acid sequencenot found in nature, which is derived by replacement of the trypsinand/or chymotrypsin loop of a precursor BBPI and by replacement of atleast one amino acid residue of a precursor BBPI with a different aminoacid. In some embodiments, the substitution of the at least one aminoacid generates a modified variant BBPI that has a greater proteaseinhibitory activity than that of the unmodified variant precursor BBPI.In other embodiments, substitution of the at least one amino acidgenerates a modified variant BBPI that has a greater protease inhibitoryactivity and production yield than that of the unmodified variantprecursor BBPI.

Thus, a modified variant BBPI is derived by substituting at least oneamino acid in the backbone of any one variant BBPI scaffold as recitedherein. In some embodiments, the isolated modified variant Bowman BirkProtease Inhibitor (BBPI) contains a variant peptide that replaces thechymotrypsin loop of the BBPI scaffold and further comprises asubstituted amino acid at least at one amino acid position chosen frompositions equivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50,52, 55, and 65 of the variant BBI of SEQ ID NO:187. In otherembodiments, the isolated modified variant Bowman Birk ProteaseInhibitor (BBPI) contains a variant peptide that replaces the trypsinloop of the BBPI scaffold and further comprises a substituted amino acidat least at one amino acid position chosen from positions equivalent to1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of thevariant BBI of SEQ ID NO:187. In yet other embodiments, the isolatedmodified variant Bowman Birk Protease Inhibitor (BBPI) contains avariant peptide that replaces the trypsin and the chymotrypsin loop ofthe BBPI scaffold and further comprises a substituted amino acid atleast at one amino acid position chosen from positions equivalent to 1,4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of thevariant BBI of SEQ ID NO:187. In some embodiments, the BBPI scaffoldchosen from the scaffolds of the soybean inhibitor from Glycine max(BBI; SEQ ID NO:13) or the mature and truncated form thereof (SEQ IDNO:185), the inhibitor from Dolichos biflorus (BBdb; SEQ ID NO:449), thesoybean inhibitor D-II from Glycine max (BBsb3; SEQ ID NO:450), theinhibitor from Torresea (Amburana) cearensis (BBtc; SEQ ID NO:451), theBBI-AV scaffold of (SEQ ID NO:186), the BBIt-AV scaffold of (SEQ IDNO:187), the BBdb-AV scaffold of (SEQ ID NO:452), the BBsb3-AV scaffoldof (SEQ ID NO:453), the BBtc-AV scaffold of (SEQ ID NO:454), theBBIt-VEGK scaffold of (SEQ ID NO:640), the BBIt-VEGT scaffold of (SEQ IDNO:641) and the BBIt-VEGKD scaffold of (SEQ ID NO:642). In someembodiments, the variant peptide comprised in the modified variant BBPIis chosen from a VEGF-binding peptide, an FGF-5-binding peptide, aTGFβ-binding peptide and a TNFα-binding peptide. In some embodiments,the VEGF-binding sequences include, but are not limited to VEGF-bindingpeptides disclosed in U.S. application Ser. Nos. 09/832,723 and10/984,270, including peptides ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ IDNO:458), TLWKSYW (SEQ ID NO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ IDNO:468) KTNPSGS (SEQ ID NO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ IDNO:471) KPSSASS (SEQ ID NO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQID NO:492), PYKASFY (SEQ ID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT(SEQ ID NO:495), NPLHTLS (SEQ ID NO:496), KHERIWS (SEQ ID NO:497),ATNPPPM (SEQ ID NO:498), STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ IDNO:500), PKADSKQ (SEQ ID NO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQID NO:503), ALSAPYS (SEQ ID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY(SEQ ID NO:506), KWIVSET (SEQ ID NO:507), PNANAPS (SEQ ID NO:508),NVQSLPL (SEQ ID NO:509), TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ IDNO:511), SLWPAFW (SEQ ID NO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQID NO:514), APWNLHI (SEQ ID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY(SEQ ID NO:517), TLYPQFW (SEQ ID NO:518), and HLAPSAV (SEQ ID NO:519).In some other embodiments, the VEGF variant sequences include, but arenot limited to VEGF-binding peptides disclosed in U.S. application Ser.No. 11/919,717, including peptides KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK(SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQ ID NO:226),TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ IDNO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW(SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536).

In other embodiments, the FGF5-binding sequences include, but are notlimited to FGF5-binding peptides disclosed in U.S. application Ser. Nos.10/984,410 and 12/033,848, including peptides CACRTQPYPLCF (MM007; SEQID NO:430), CICTWIDSTPC(PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537),CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ IDNO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC(SEQ ID NO:543), TNIDSTP (SEQ ID NO:544), HLQTTET (SEQ ID NO:545),SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ IDNO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP(SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556),PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ IDNO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671).

In other embodiments, the variant peptide is a TGF-β-binding peptide ischosen from TGFβ-binding sequences that include, but are not limited toTGFβ-binding peptides disclosed in U.S. application Ser. No. 10/581,142,including peptides CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF(MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC(SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565),QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ IDNO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568),acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570),NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ IDNO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ(SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581),ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ IDNO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC(SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673) andWTQHIHNC (SEQ ID NO:674).

In yet other embodiments, the variant peptide is a TNFα-binding peptideis chosen from TNFα binding sequences that include, but are not limitedto TNF-binding peptides disclosed in U.S. application Ser. No.10/968,732, including peptides RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2;SEQ ID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583),ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ IDNO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH(SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).

In some embodiments, the at least one amino acid substitution containedat least at one equivalent to a position chosen from positionsequivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50,52, 55, and 65 of SEQ ID NO:187 result in the following substitutedamino acids. In one embodiment, the substituted amino acid at the aminoacid position equivalent to position 1 of SEQ ID NO:187 is chosen from Aand C. In another embodiment, the substituted amino acid at the aminoacid position equivalent to position 4 of SEQ ID NO:187 is V. In anotherembodiment, the substituted amino acid at the amino acid positionequivalent to position 5 of SEQ ID NO:187 is chosen from P, and A. Inanother embodiment, the substituted amino acid at the amino acidposition equivalent to position 11 of SEQ ID NO:187 is G. In anotherembodiment, the substituted amino acid at the amino acid positionequivalent to position 13 of SEQ ID NO:187 is chosen from Y, I, F, M, L,V, K, and R. In another embodiment, the substituted amino acid at theamino acid position equivalent to position 18 of SEQ ID NO:187 include1, V and L. In another embodiment, the substituted amino acid at theamino acid position equivalent to position 25 of SEQ ID NO:187 is chosenfrom K, N, W, I, A and R. In another embodiment, the substituted aminoacid at the amino acid position equivalent to position 27 of SEQ IDNO:187 include R, K, V, A, and Q. In another embodiment, the substitutedamino acid at the amino acid position equivalent to position 29 of SEQID NO:187 is chosen from R, K, and P. In another embodiment, thesubstituted amino acid at the amino acid position equivalent to position31 of SEQ ID NO:187 is chosen from Q, H, E, A, R, W, K and T. In anotherembodiment, the substituted amino acid at the amino acid positionequivalent to position 38 of SEQ ID NO:187 is chosen from N, K and R. Inanother embodiment, the substituted amino acid at the amino acidposition equivalent to position 40 of SEQ ID NO:187 is chosen from H, K,Q, R, and Y. In another embodiment, the substituted amino acid at theamino acid position equivalent to position 50 of SEQ ID NO:187 is chosenfrom R, Q, K, T, V, M, and S. In another embodiment, the substitutedamino acid at the amino acid position equivalent to position 52 of SEQID NO:187 is chosen from K, T, R, Q, L, H, A, M, S and E. In anotherembodiment, the substituted amino acid at the amino acid positionequivalent to position 55 of SEQ ID NO:187 is M. In another embodiment,the substituted amino acid at the amino acid position equivalent toposition 65 of SEQ ID NO:187 is chosen from E, Q, and D. In someembodiments, a single amino acid substitution made in a variant BBPIresults in a modified variant BBPI that has a greater proteaseinhibitory activity than that of the precursor unmodified variant BBPI.In some embodiments, a single amino acid substitution generates amodified variant BBPI that has greater trypsin inhibitory activity (TIA)than the unmodified precursor variant BBPI; while in other embodiments,a single amino acid substitution generates a modified variant BBPI thathas greater chymotrypsin inhibitory activity (CIA) than the unmodifiedprecursor variant BBPI.

In one embodiment, the modified variant BBPI is the variant BBI of SEQID NO:187 (BBIt-AV; FIG. 9), which contains a VEGF variant peptide inplace of the chymotrypsin loop, and which is further modified to containan amino acid substitution at least at one position chosen frompositions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and65 of SEQ ID NO:187 to generate a modified variant BBI. Any one of thesingle amino acid substitutions described above and made in the variantBBIt-AV BBPI of SEQ ID NO:187 generated modified variant BBIt-AV BBPIsthat have greater trypsin inhibitory activity than the unmodifiedprecursor variant BBIt-AV (SEQ ID NO:187; see Example 10).

5.4.2 Modified Variant BBPIs: Combinations of Amino Acid Substitutions

The invention encompasses modified variant BBPIs comprising at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten, at leasteleven, at least twelve, at least thirteen, at least fourteen, at leastfifteen and at least sixteen amino acid substitutions. In someembodiments, the at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight amino, at least nine,at least ten, at least eleven, at least twelve, at least thirteen, atleast fourteen, at least fifteen and at least sixteen acid substitutionsgenerate modified variant BBPIs that have greater TIA than theunmodified precursor variant BBPI. In other embodiments, the at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, amino acid substitutions generate modifiedvariant BBPIs that have greater TIA and production yield than theunmodified precursor variant BBPI.

In some embodiments, the modified variant BBPI comprises a combinationof two amino acid substitutions at amino acids at positions equivalentto positions 50 and 52 of SEQ ID NO:187. In some embodiments, thecombination of two amino acid substitutions is 50T-52A. The inventionprovides for any one of the variant BBPI scaffolds described in Section5.3 and further comprising the combination of the two amino acidsubstitutions 50T-52A, as described in section 5.4. In one embodiment,the chymotrypsin loop of the variant scaffold is a VEGF variant peptidee.g. SEQ ID NO:9, and the variant scaffold is altered further tocomprise a combination of three amino acid substitutions at positionsequivalent to positions 13, 50 and 52 of SEQ ID NO:187 to generate amodified variant BBPI scaffold. In one embodiment, the modified variantBBPI comprising a combination of two amino acid substitutions is themodified variant BBIt-AV-F50T-V52A of SEQ ID NO: 595(DPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:595).

In some embodiments, the modified variant BBPI comprises a combinationof three amino acid substitutions at amino acids at positions equivalentto positions 13, 50 and 52 of SEQ ID NO:187. In some embodiments, thecombination of three amino acid substitutions is chosen from acombination of substitutions at positions 25-50-52, 29-50-52, 40-50-52,and 13-50-52. In some embodiments, the combination of three amino acidsubstitutions is chosen from 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and13I-50T-52A. The invention provides for any one of the variant BBPIscaffolds described in Section 5.3 and further comprising thecombination of the three amino acid substitutions chosen from25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A, as described insection 5.4. In one embodiment, the chymotrypsin loop of the variantscaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variantscaffold is altered further to comprise a combination of three aminoacid substitutions at positions equivalent to positions 13, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold.

In one embodiment, the modified variant BBPI comprising a combination ofthree amino acid substitutions is chosen from the modified variantBBIt-AV-S25L-F50T-V52A of SEQ ID NO: 603(DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:603), the modified variant BBIt-AV-L29P-F50T-V52A of SEQ IDNO:607(DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:607), and the modified variant BBIt-AV-A40K-F50T-V52A of SEQID NO:609(DPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:609).

In some embodiments, the modified variant BBPI comprises a combinationof four amino acid substitutions at amino acids at positions equivalentto positions 13, 29, 50 and 52 of SEQ ID NO:187. In some embodiments,the combination of four amino acid substitutions is chosen from acombination of substitutions at positions 13-25-50-52, 13-29-50-52,25-29-50-52, 13-40-50-52, 25-40-50-52, and 29-40-50-52. In someembodiments, the combination of four amino acid substitutions is chosenfrom 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A,25L-40K-50T-52A, and 29P-40K-50T-52A. The invention provides for any oneof the variant BBPI scaffolds described in Section 5.3 and furthercomprising the combination of the four amino acid substitutions chosenfrom 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A, 13I-40K-50T-52A,25L-40K-50T-52A, and 29P-40K-50T-52A, as described in section 5.4. Inone embodiment, the chymotrypsin loop of the variant scaffold is a VEGFvariant peptide chosen from SEQ ID NO:9 and 460, and the variantscaffold is altered further to comprise a combination of four amino acidsubstitutions at positions equivalent to positions 13, 29, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of fouramino acid substitutions is chosen from the modified variantBBIt-AV-A13I-S25L-F50T-V52A of SEQ ID NO:596(DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:596), the modified variant BBIt-AV-A13I-L29P-F50T-V52A of SEQID NO:600(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:600), the modified variant BBIt-AV-A13I-A40K-F50T-V52A of SEQID NO:602(DPDDESSKPCCDQCICTKSNPPQCRCSDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:602), the modified variant BBIt-AV-S25L-L29P-F50T-V52A of SEQID NO:604(DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:604), the modified variant BBIt-AV-S25L-A40K-F50T-V52A of SEQID NO:606(DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:606), the modified variant BBIt-AV-L29P-A40K-F50T-V52A of SEQID NO:608(DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:608), and the modified variant BBIt-VEGKD-A13I-S25K-L29P-V52Kof SEQ ID NO:643 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C KD M R P N S C H S A C K S C I C K Y D L Y W W C F C K D I T D F C Y E PC K P S E; SEQ ID NO:643). In another embodiment, the chymotrypsin loopof the variant scaffold is an FGF5 variant peptide chosen from SEQ IDNOS:430 and 431, and the variant scaffold is altered further to comprisea combination of four amino acid substitutions at positions equivalentto positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modifiedvariant BBPI scaffold. In one embodiment, the modified variant BBPIcomprising a combination of four amino acid substitutions is chosen fromthe modified variant BBIt-MM007-Q-A13I-L29P-F50T-V52A of SEQ ID NO:432,and the modified variant BBIt-FGFps2-Q-A13I-L29P-F50T-V52A of SEQ IDNO:434. In another embodiment, the chymotrypsin loop of the variantscaffold is a TGFβ variant peptide chosen from SEQ ID NOS:436, 437, 438,672, 673, and 674 and the variant scaffold is altered further tocomprise a combination of four amino acid substitutions at positionsequivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187 to generate amodified variant BBPI scaffold. In one embodiment, the modified variantBBPI comprising a combination of four amino acid substitutions is chosenfrom the modified variant BBIt-PEN3-Q-A13I-L29P-F50T-V52A of SEQ IDNO:443, the modified variant BBIt-MM021W-Q-A13I-L29P-F50T-V52A of SEQ IDNO:445, and the modified variant BBIt-WTQ-Q-A13I-L29P-F50T-V52A of SEQID NO:447.

In some embodiments, the modified variant BBPI comprises a combinationof five amino acid substitutions at amino acids at positions equivalentto positions 13, 29, 40, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of five amino acid substitutions is chosenfrom a combination of substitutions at positions 13-25-29-50-52,13-29-40-50-52, 13-25-40-50-52, 25-29-40-50-52, and 13-29-40-50-52. Insome embodiments, the combination of five amino acid substitutions ischosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A,13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A,13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T. Theinvention provides for any one of the variant BBPI scaffolds describedin Section 5.3 and further comprising the combination of the five aminoacid substitutions chosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A,13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A,13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T, asdescribed in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and thevariant scaffold is altered further to comprise a combination of fiveamino acid substitutions at positions equivalent to positions 13, 29,40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPIscaffold. In one embodiment, the modified variant BBPI comprising acombination of five amino acid substitutions is chosen from the modifiedvariant BBIt-AV-A13I-S25L-L29P-F50T-V52A of SEQ ID NO:597(DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:597), the modified variant BBIt-AV-A13I-L29P-A40K-F50T-V52A ofSEQ ID NO:599(DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:599), the modified variant BBIt-AV-A13I-S25L-A40K-F50T-V52A ofSEQ ID NO:601(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:601), the modified variant BBIt-AV-S25L-L29P-A40K-F50T-V52A ofSEQ ID NO:605(DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:605), the modified variant BBIt-AV-A13L-L29P-A40K-F50T-V52A ofSEQ ID NO:615(DPDDESSKPCCDQCLCTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:615), the modified variant BBIt-AV-A13I-L29K-A40K-F50T-V52A ofSEQ ID NO:620(DPDDESSKPCCDQCICTKSNPPQCRCSDMRKNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:620), the modified variant BBIt-AV-A13I-L29P-A40K-F50K-V52A ofSEQ ID NO:624(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCKCADITDFCYEPCKPSE;SEQ ID NO:624), and the modified variantBBIt-AV-A13I-L29P-A40K-F50T-V52T of SEQ ID NO:625(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCTDITDFCYEPCKPSE;SEQ ID NO:625).

In some embodiments, the modified variant BBPI comprises a combinationof six amino acid substitutions at amino acids at positions equivalentto positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of six amino acid substitutions is chosenfrom a combination of substitutions at positions 13-25-29-40-50-52,1-13-29-40-50-52, 4-13-29-40-50-52, 5-13-29-40-50-52, 11-13-29-40-50-52,13-25-29-40-50-52, 13-27-29-40-50-52, 13-29-31-40-50-52,13-29-31-40-50-52, 13-29-38-40-50-52, and 13-29-38-40-50-52. In someembodiments, the combination of six amino acid substitutions is chosenfrom 13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A,4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A,13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A,13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A,13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A. The inventionprovides for any one of the variant BBPI scaffolds described in Section5.3 and further comprising the combination of the six amino acidsubstitutions chosen from 13I-25L-29P-40K-50T-52A,1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A,11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A,13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A,13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and13I-29P-38N-40K-50T-52A, as described in section 5.4. In one embodiment,the chymotrypsin loop of the variant scaffold is a VEGF variant peptideof SEQ ID NO:9, and the variant scaffold is altered further to comprisea combination of six amino acid substitutions at positions equivalent topositions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187 to generate amodified variant BBPI scaffold. In one embodiment, the modified variantBBPI comprising a combination of six amino acid substitutions is chosenfrom the modified variant BBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A of SEQID NO:598(DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:598), the modified variantBBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:611(DPDDEVSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:611), the modified variantBBIt-AV-S4V-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:612(DPDDEVSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:612), the modified variantBBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:613(DPDDESPKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:613), the modified variantBBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:614(DPDDESSKPCCDGCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:614), the modified variantBBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A- of SEQ ID NO:616(DPDDESSKPCCDQCICTKSNPPQCRCRDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:616), the modified variantBBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A of SEQ ID NO:619(DPDDESSKPCCDQCICTKSNPPQCRCSDRRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:619), the modified variantBBIt-AV-A13I-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:621(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNACHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:621), the modified variantBBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:622(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNRCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:622), the modified variantBBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:623(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKNCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:623), and the modified variantBBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:626(DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPEE;SEQ ID NO:626).

In some embodiments, the modified variant BBPI comprises a combinationof seven amino acid substitutions at amino acids at positions equivalentto positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of seven amino acid substitutions is chosenfrom a combination of substitutions at positions 13-25-29-31-40-50-52,13-25-29-31-40-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52, and13-25-27-29-31-50-52. In some embodiments, the combination of sevenamino acid substitutions is chosen from 13L-25R-29P-31A-40K-50T-52A,13L-25R-29P-31R-40K-50T-52A, and 13I-25R-27A-29P-31A-50K-52T. Theinvention provides for any one of the variant BBPI scaffolds describedin Section 5.3 and further comprising the combination of the six aminoacid substitutions chosen from 13L-25R-29P-31A-40K-50T-52A,13L-25R-29P-31R-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, and 13I-25R-27A-29P-31A-50K-52T, asdescribed in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and thevariant scaffold is altered further to comprise a combination of sevenamino acid substitutions at positions equivalent to positions 13, 25,29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modified variantBBPI scaffold. In one embodiment, the modified variant BBPI comprising acombination of seven amino acid substitutions is chosen from themodified variant BBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A of SEQ IDNO:617(DPDDESSKPCCDQCICTKSNPPQCRCRDMRPNACHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:617), the modified variantBBIt-AV-A13I-S25R-L29P-S31R-A40K-F50T-V52A of SEQ ID NO:618(DPDDESSKPCCDQCICTKSNPPQCRCRDMRPNRCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:618), the modified variant ofBBIt-VEGF-V1-A13I-S25R-M27A-L29P-S31A-F50K-V52T SEQ ID NO:491 (D P D D ES S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C K S CA C S K H S Q I T C K C T D I T D F C Y E P C K P S E; SEQ ID NO:491),the modified variant BBIt-VEGF-V2-A13I-S25R-M27A-L29P-S31A-F50K-V52T ofSEQ ID NO:632 (D P D D E S S K P C C D Q C I C T K S N P P Q C R C R D AR P N A C H S A C K S C A C K T N P S G S C K C T D I T D F C Y E P C KP S E: SEQ ID NO:632), the modified variantBBIt-VEGF-V3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:633 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C KS C A C R P T G H S L C K C T D I T D F C Y E P C K P S E; SEQ IDNO:633), the modified variantBBIt-VEGF-V4-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:634 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C KS C A C K H S A K A E C K C T D I T D F C Y E P C K P S E; SEQ IDNO:634), the modified variantBBIt-VEGF-V5-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:635 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C KS C A C K P S S A S S C K C T D I T D F C Y E P C K P S E; SEQ IDNO:635), the modified variantBBIt-VEGF-V6-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:636 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C KS C A C P V T K R V H C K C T D I T D F C Y E P C K P S E; SEQ IDNO:636), the modified variantBBIt-TNFα-T1-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:637 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C KS C A C R Y W Q D I P C K C T D I T D F C Y E P C K P S E; SEQ IDNO:637), the modified variantBBIt-TNFα-T2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:638 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C KS C A C A P E P I L A C K C T D I T D F C Y E P C K P S E; SEQ IDNO:638), and the modified variantBBIt-TNFα-T3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:639(DPDDESSKPCCDQCICTKSNPPQCRCKDQRPNECHSACKSCHCYNLYGWTCRCQDITDFCYEPCKPPE;SEQ ID NO:639).

In some embodiments, the modified variant BBPI comprises a combinationof eight amino acid substitutions at amino acids at positions equivalentto positions 13, 25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of eight amino acid substitutions is chosenfrom a combination of substitutions at positions13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52,13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, and13-25-27-29-31-40-50-52. In some embodiments, the combination of eightamino acid substitutions is chosen from combinations13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q,13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and13I-25K-27Q-29P-31E-40H-50R-52Q. The invention provides for any one ofthe variant BBPI scaffolds described in Section 5.3 and furthercomprising the combination of the eight amino acid substitutions chosenfrom combinations 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K,13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q, asdescribed in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and thevariant scaffold is altered further to comprise a combination of sevenamino acid substitutions at positions equivalent to positions 13, 25,27, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modifiedvariant BBPI scaffold. In one embodiment, the modified variant BBPIcomprising a combination of eight amino acid substitutions is chosenfrom the modified variantBBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T of SEQ ID NO:627 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C R D A R P N A C H S A C KS C H C Y N L Y G W T C K C T D I T D F C Y E P C K P S E; SEQ IDNO:627; KT8), the modified variant ofBBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q of SEQ ID NO:628 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C K D A R R N E C H S A C KS C K C Y N L Y G W T C Q C Q D I T D F C Y E P C K P S E; SEQ IDNO:628; QQ8), the modified variantBBIt-AV-A13I-S25K-M27R-L29E-S31A-A40H-F50R-V52K of SEQ ID NO:629 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C K D R R E N A C H S A C KS C H C Y N L Y G W T C R C K D I T D F C Y E P C K P S E; SEQ IDNO:629; RK8), the modified variantBBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L of SEQ ID NO:630 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C K D A R R N A C H S A C KS C H C Y N L Y G W T C R C L D I T D F C Y E P C K P S E; SEQ ID NO:630; RL8) and the modified variantBBIt-AV-A13I-S25K-M27Q-L29P-S31E-A40H-F50R-V52Q of SEQ ID NO:631 (D P DD E S S K P C C D Q C I C T K S N P P Q C R C K D Q R P N E C H S A C KS C H C Y N L Y G W T C R C Q D I T D F C Y E P C K P S E; SEQ IDNO:631; RQ8).

The invention further provides for modified variant BBPIs that compriseany one combination of the amino acid substitutions described above andthat have greater protease inhibitory activity than the unmodifiedprecursor variant BBPI. In some embodiments, modified variant BBPIswhich contain a variant peptide in place of the chymotrypsin loop of thecorresponding precursor unmodified BBPI have greater trypsin inhibitoryactivity (TIA) than that of the precursor unmodified BBPI scaffold. Inother embodiments, modified variant BBPIs which contain a variantpeptide in place of the trypsin loop of the corresponding precursorunmodified BBPI have greater chymotrypsin inhibitory activity (TIA) thanthat of the precursor unmodified BBPI scaffold.

As shown in the Examples, substitutions of at least one amino acid inthe backbone of the variant BBPI generates a modified variant BBPI thathas a greater production yield that the unmodified variant BBPI. In someembodiments, BBPIs that comprise a combination of two, three, four,five, six, seven or eight amino acid substitutions have a greaterproduction yield than the unmodified precursor BBPI. Thus, the inventionprovides for modified variant BBPIs that comprise any one combination ofthe amino acid substitutions described above and that have greaterproduction yield (PY) than the unmodified precursor variant BBPIs. Inyet other embodiments, the invention provides for modified variant BBPIsthat comprise any one combination of the amino acid substitutionsdescribed above and that have greater trypsin inhibitory activity andgreater production yield than the TIA and PY of the unmodified precursorvariant BBPIs.

In some embodiments, the modified variant BBPIs further comprise apeptide insert that is positioned at the N-terminus of the modifiedvariant BBPI. In some embodiments, the peptide insert comprises asequence of between 1 and 15 amino acids. In other embodiments, thepeptide insert comprises a sequence between 5 and 10 amino acids. Insome embodiments, the peptide insert comprises the peptide of SEQ IDNO:389 (DDEPSKPCCDPDP; SEQ ID NO:389). Examples of modified variantBBPIs that the peptide insert of SEQ ID NO:389 are the modified variant4D13BBIt-AV of(DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE;SEQ ID NO:390), and the modified variantBBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413(DPDDEPSKPCCDPDPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE;SEQ ID NO:413).

5.4.3 BBPI Fusion Proteins

In some embodiments, each modified variant BBPI is expressed as fusionprotein comprising a catalytic domain, a cleavage site and the BBPIscaffold. The catalytic domain is chosen from cellulase, cutinase, anddisulfide isomerase. In some embodiments, the catalytic domain comprisedin the BBPI fusion protein is the cellulase catalytic domain of SEQ IDNO:669

(SEQ ID NO: 669) DDYSVVEEHGQLSISNGELVNERGEQVQLKGMSSHGLQWYGQFVNYESMKWLRDDWGITVFRAAMYTSSGGYIDDPSVKEKVKETVEAAIDLGIYVIIDWHILSDNDPNIYKEEAKDFFDEMSELYGDYPNVIYEIANEPNGSDVTWDNQIKPYAEEVIPVIRDNDPNNIVIVGTGTWSQDVHHAADNQLADPNVMYAFHFYAGTHGQNLRDQVDYALGQGAAIFVSEWGTSAATGDGGVFLDEAQVWIDFMDERNLSWANWSLTHKDESSAALMPGANPTGGWTEAELSPSGTFVREKI RESAS

The fusion protein is processed by a protease or acid/heat treatment toliberate the modified variant BBPI. In some embodiments, the fusionprotein further comprises at least one linker sequence. In someembodiments, the linker sequence is selected from the group consistingof SEQ ID NOS:141-143. Although cleavage of the fusion polypeptide torelease the modified variant BBPI will often be useful, it is notnecessary. Modified variant BBPIs expressed and secreted as fusionproteins surprisingly retain their function.

The modified variant BBPI fusion proteins are each expressed by the hostbacterial cell from a fusion polynucleotide sequence. Such fusionpolynucleotide sequences are assembled in proper reading frame from the5′ terminus to 3′ terminus in the order of first, second, third andfourth polynucleotide sequences. As so assembled, the polynucleotidesequence encodes a “fusion polypeptide” encoding from itsamino-terminus 1. a signal peptide functional as a secretory sequence ina bacterial species, 2. a secreted polypeptide or portion thereofnormally secreted from a bacterial species e.g. cellulase or portionthereof, 3. a cleavable linker peptide and a 4. desired polypeptide(e.g., a modified variant BBPI). In some embodiments, the above-definedfusion polynucleotide sequence further comprises a polynucleotideencoding a portion of a propeptide that functions as a spacer betweenthe first and second polynucleotide sequences of a fusion protein. Thefunction of the spacer is intended to increase the distance between thefirst and second encoded polypeptides. In some embodiments, the spacersequence is 1-10 amino acids long. In other embodiments, theabove-defined fusion polynucleotide sequence further comprises apolynucleotide encoding a peptide insert between the linker peptide andthe modified variant BBPI. In some embodiments, the peptide insertcomprises a sequence of between 1 and 15 amino acids. In otherembodiments, the peptide insert comprises a sequence between 5 and 10amino acids. In some embodiments, the peptide insert comprises thepeptide of SEQ ID NO:389.

Various methods are known to those in the art for the production offusion proteins (See e.g., U.S. Pat. Nos. 5,411,873, 5,429,950, and5,679,543, all of which are incorporated by reference herein). Thus, itis intended that any suitable method will find use in the presentinvention.

5.5 Bowman Birk Protease Inhibitor Polynucleotides

To the extent that the present invention depends on the production offusion proteins, it relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory Manual ((2nd ed.) [1989]); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Ausubel et al., (eds.),Current Protocols in Molecular Biology (1994).

The invention provides for compositions including polynucleotideconstructs, vectors and host cells that enable the expression ofmodified variant BBPIs. The polynucleotide constructs of the inventioncomprise a promoter sequence and a fusion polynucleotide sequence thatencodes a fusion protein comprising a modified variant BBPI. Asdescribed above, the fusion polynucleotide sequence comprises acatalytic domain, a cleavage site and the BBPI scaffold. Natural orsynthetic polynucleotide fragments encoding a BBPI may be incorporatedinto the polynucleotide constructs. The at least one amino acidsubstitution introduced into a variant BBPI is generated by means ofsite saturation mutagenesis in at least one codon. In alternativeembodiments, the at least one amino acid substitution is encoded by DNAoligonucleotides that contain the encoding sequence, and that areannealed and ligated into the protease inhibitor DNA sequence. Thedesired DNA sequence is then isolated and used in the methods providedherein.

In some embodiments, the polynucleotide constructs of the inventioncomprise polynucleotide sequences that encode a modified variant BBPIthat shares at least about 65% amino acid sequence identity, at leastabout 70% amino acid sequence identity, at least about 75% amino acidsequence identity, at least about 80% amino acid sequence identity, atleast about 85% amino acid sequence identity, at least about 90% aminoacid sequence identity, at least about 92% amino acid sequence identity,at least about 95% amino acid sequence identity, at least about 97%amino acid sequence identity, at least about 98% amino acid sequenceidentity, and at least about 99% amino acid sequence identity with theamino acid sequence of the unmodified precursor variant BBPI and hasgreater protease inhibitory activity than the unmodified precursorvariant BBPI. The invention further provides for polynucleotidesencoding modified variant BBPIs that comprise any one combination of theamino acid substitutions described above and that have greater proteaseinhibitory activity e.g. trypsin inhibitory activity, than theunmodified precursor variant BBPI.

In some embodiments, the polynucleotide constructs of the inventioncomprise a polynucleotide sequence that may be codon optimized forexpression of a modified variant BBPI in the host cell used. Since codonusage tables listing the usage of each codon in many cells are known inthe art (See, e.g., Nakamura et al., Nucl. Acids Res., 28:292 [2000]) orreadily derivable, such nucleic acids can be readily designed giving theamino acid sequence of a protein to be expressed.

The invention also encompasses polynucleotide constructs that comprisecoding sequences encoding modified variant BBPI proteins that arerelated by being structurally and/or functionally similar. In someembodiments, a modified variant BBPI is derived from anaturally-occurring BBPI belonging to a different genus and/or species.In some embodiments, related proteins are provided from the samespecies. Indeed, it is not intended that the present invention belimited to related proteins from any particular source(s). In addition,the term “related proteins” encompasses tertiary structural homologs andprimary sequence homologs. For example, the present inventionencompasses such homologues including but not limited to such BBPIproteins such as those described by Prakash et al. (J mol Evol42:560-569 [1996]).

In some embodiments, the promoter sequence comprised in thepolynucleotide constructs of the invention is operably linked to theBBPI-encoding polynucleotide. Exemplary promoters include bothconstitutive promoters and inducible promoters. Such promoters are wellknown to those of skill in the art. Those skilled in the art are alsoaware that a natural promoter can be modified by replacement,substitution, addition or elimination of one or more nucleotides withoutchanging its function. The practice of the present invention encompassesand is not constrained by such alterations to the promoter. The choiceof promoter used in the genetic construct is within the knowledge of oneskilled in the art.

In some embodiments, the promoter sequence may be obtained from abacterial source. In some embodiments, the promoter sequence may beobtained from a Gram-positive bacterium such as a Bacillus strain (e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, or Bacillus thuringiensis); or a Streptomyces strain(e.g., Streptomyces lividans or Streptomyces murinus); or from a gramnegative bacterium (e.g., E. coli or Pseudomonas sp.).

The promoter can be any DNA sequence having transcription activity inthe host organism of choice and can be derived from genes that arehomologous or heterologous to the host organism. Examples of suitablepromoters that can be used to express a modified variant BBPI in abacterial host include, but are not limited to the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), the aprE promoter of Bacillus subtilis, the promoters of theBacillus stearothermophilus maltogenic amylase gene (amyM), thepromoters of the Bacillus amyloliquefaciens α-amylase gene (amyQ), thepromoters of the Bacillus subtilis xylA and xylB genes and a promoterderived from a Lactococcus sp.-derived promoter including the PI70promoter. When the gene encoding the compound is expressed in abacterial species such as E. coli, a suitable promoter can be selected,for example, from a bacteriophage promoter including a T7 promoter and aphage lambda promoter. For transcription in a fungal species, examplesof useful promoters are those derived from the genes encoding theAspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,A. niger neutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase, and A. nidulans acetamidase. Examplesof suitable promoters for the expression in a yeast species include butare not limited to the Gal 1 and Gal 10 promoters of Saccharomycescerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.

The invention also encompasses promoter sequences that have been mutatedto increase the activity of the promoter when compared to the activityof the corresponding wild-type promoter resulting in the expression ofthe modified variant BBPI protein. Thus, it is understood that variantsof the sequences that define the B. subtilis AprE promoter find use inthe constructs of the invention. Methods for creating promoter variantsin Bacillus sp. are well known in the art (See e.g., Helmann et al.,2002. RNA polymerase and sigma factors, pp 289-312 In A. L. Sonenshein,J. A. Hoch and R. Losick (ed), Bacillus subtilis and its closestrelatives: from genes to cells. American Society for Microbiology,Washington, D.C.) It is not intended that the present invention belimited to any particular promoter, as any suitable promoter known tothose skilled in the art finds use with the present invention.

In embodiments, in addition to a promoter sequence, the polynucleotideconstruct also contains a transcription termination region downstream ofthe structural gene to provide for efficient termination. In someembodiments, the termination region is obtained from the same gene asthe promoter sequence, while in other embodiments it is obtained fromanother gene. The selection of suitable transcription terminationsignals is well-known to those of skill in the art.

5.6 Bowman Birk Protease Inhibitor Vectors

The invention provides vectors comprising the polynucleotide constructsof the invention. The vectors are introduced into a host cell to expressthe modified variant BBPI proteins of the invention. Any vector may beused as long as it is replicable and viable in the cells into which itis introduced. Large numbers of suitable vectors and promoters are knownto those of skill in the art, and are commercially available.Appropriate cloning and expression vectors are also described in variousreferences known to those in the art (See e.g., Sambrook et al., supraand Ausubel et al., supra, expressly incorporated by reference herein).The appropriate BBPI-encoding DNA sequence is inserted into a plasmid orvector (collectively referred to herein as “vectors”) by any suitablemethod. In general, the DNA sequence is inserted into an appropriaterestriction endonuclease site(s) by standard procedures known to thosein the art.

Appropriate vectors are typically equipped with a selectablemarker-encoding nucleic acid sequence, insertion sites, and suitablecontrol elements, such as termination sequences. In some embodiments,the vectors comprise regulatory sequences, including, for example,control elements (i.e., promoter and terminator elements or 5′ and/or 3′untranslated regions), effective for expression of the coding sequencein host cells (and/or in a vector or host cell environment in which amodified soluble protein coding sequence is not normally expressed),operably linked to the coding sequence. Large numbers of suitablevectors and promoters are known to those of skill in the art, many ofwhich are commercially available and known to those in the art. Thechoice of the proper selectable marker will depend on the host cell.Appropriate markers for different bacterial hosts are well known in theart. Typical selectable marker genes encode proteins that (a) conferresistance to antibiotics or other toxins (e.g., ampicillin,methotrexate, tetracycline, neomycin mycophenolic acid, puromycin,zeomycin, or hygromycin; or (b) complement an auxotrophic mutation or anaturally occurring nutritional deficiency in the host strain.

In some embodiments, expression of a fusion BBPI polypeptide resultsfrom the expression of one or more copies of the corresponding fusionpolypeptide-encoding polynucleotide that is present on amulticopy/replicating plasmid that has been introduced into a host cell.In some embodiments, the vector is a multicopy/replicating plasmidvector which forms an extrachromosomal self-replicating genetic elementthat expresses a fusion BBPI protein in the host cell. Typically, thevector is a plasmid vector, which carries a selectable marker gene thatallows for ease of selecting the host cells that contain the plasmid.Vectors that replicate autonomously in a host cell include vectors thatcomprise an origin of replication, which enables the vector to replicateautonomously in the Bacillus cell. Examples of bacterial origins ofreplication are the origins of replication of plasmids pBR322, pUC19,pACYC177, and pACYC184 permitting replication in E. coli, and pUB110,pC194, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. Theorigin of replication may be one having a mutation to make its functiontemperature-sensitive in the Bacillus cell (See, e.g., Ehrlich,Proceedings of the National Academy of Sciences USA 75:1433 [1978]).Additional bacterial expression vectors that find use in the presentinvention include bacteriophages λ and M13, and fusion expressionsystems such as MBP, GST, and LaZ. In further embodiments, epitope tagsare added to recombinant proteins, in order to provide convenientmethods of isolation (e.g., c-myc).

In some embodiments, the expression of a BBPI fusion polypeptide resultsfrom the expression of at least one copy of a BBPI fusion-encodingpolynucleotide that is integrated into the genome of the host cell.Thus, in some embodiments, the invention provides a BBPI encodingpolynucleotide construct that is incorporated into an integratingvector. Thus, when the vector is introduced into the host cell, it isintegrated into the genome and replicated together with the genome intowhich it has integrated. Multiple copies of the BBPI gene can beintegrated at several positions in the genome of the host cell.Alternatively, an amplifiable expression cassette carrying a sequenceencoding a BBPI fusion protein and a selectable marker (e.g., anantimicrobial resistance marker, such as a gene coding chloramphenicolacetyl transferase) can be integrated in the genome via a singlecross-over event and then amplified by challenging the transformed hostcell with increasing concentrations of the appropriate antimicrobial(e.g., chloramphenicol).

5.7 Bowman Birk Protease Inhibitor Host Cells

In one embodiment, the invention provides for a host cell transformedwith an expression vector comprising a polynucleotide sequence encodinga modified variant BBPI. After the expression vector is introduced intothe host cells, the transformed host cells are cultured under conditionsfavoring expression of gene encoding the desired BBPI fusion protein.Large batches of transformed cells can be cultured as described above.Finally, product is recovered from the culture using techniques known inthe art.

Methods for introducing DNA into Bacillus cells involving plasmidconstructs and transformation of plasmids into bacterial host cells arewell known. In some embodiments, the plasmids are subsequently isolatedfrom E. coli and transformed into Bacillus. However, it is not essentialto use intervening microorganisms such as E. coli, and in someembodiments, a DNA construct or vector is directly introduced into aBacillus host. Those of skill in the art are well aware of suitablemethods for introducing polynucleotide sequences into Bacillus cells(See e.g., Ferrari et al., “Genetics,” in Harwood et al. (ed.),Bacillus, Plenum Publishing Corp. [1989], pages 57-72; Saunders et al.,J. Bacteriol, 157:718-726 [1984]; Hoch et al., J. Bacteriol.,93:1925-1937 [1967]; Mann et al., Current Microbiol., 13:131-135 [1986];and Holubova, Folia Microbiol., 30:97 [1985]; Chang et al., Mol. Gen.Genet., 168:11-115 [1979]; Vorobjeva et al., FEMS Microbiol. Lett.,7:261-263 [1980]; Smith et al., Appl. Env. Microbiol., 51:634 [1986];Fisher et al., Arch. Microbiol., 139:213-217 [1981]; and McDonald, J.Gen. Microbiol., 130:203 [1984]). Indeed, such methods astransformation, including protoplast transformation and congression,transduction, and protoplast fusion are known and suited for use in thepresent invention. Methods of transformation are particularly preferredto introduce a DNA construct provided by the present invention into ahost cell.

In addition to commonly used methods, in some embodiments, host cellsare directly transformed (i.e., an intermediate cell is not used toamplify, or otherwise process, the DNA construct prior to introductioninto the host cell). Introduction of the DNA construct into the hostcell includes those physical and chemical methods known in the art tointroduce DNA into a host cell without insertion into a plasmid orvector. Such methods include, but are not limited to electroporation,insertion of naked DNA or liposomes and the like. In additionalembodiments, DNA constructs are co-transformed with a plasmid, withoutbeing inserted into the plasmid. In further embodiments, a selectivemarker is deleted from the altered Bacillus strain by methods known inthe art (See, Stahl et al., J. Bacteriol., 158:411-418 [1984]; andPalmeros et al., Gene 247:255-264 [2000]).

Methods known in the art to transform Bacillus, include such methods asplasmid marker rescue transformation, which involves the uptake of adonor plasmid by competent cells carrying a partially homologousresident plasmid (Contente et al., Plasmid 2:555-571 [1979]; Haima etal., Mol. Gen. Genet., 223:185-191 [1990]; Weinrauch et al., J.Bacteriol., 154:1077-1087 [1983]; and Weinrauch et al., J. Bacteriol.,169:1205-1211 [1987]). In this method, the incoming donor plasmidrecombines with the homologous region of the resident “helper” plasmidin a process that mimics chromosomal transformation.

Other methods involving transformation by protoplast transformation arewell known in the art (See e.g., Chang and Cohen, Mol. Gen. Genet.,168:111-115 [1979]; Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263[1980]; Smith et al., Appl. Env. Microbiol., 51:634 [1986]; Fisher etal., Arch. Microbiol., 139:213-217 [1981]; McDonald [1984] J. Gen.Microbiol., 130:203 [1984]; and Bakhiet et al., 49:577 [1985]). Inaddition, Mann et al., (Mann et al., Curr. Microbiol., 13:131-135[1986]) describe transformation of Bacillus protoplasts, and Holubova(Holubova, Microbiol., 30:97 [1985]) describe methods for introducingDNA into protoplasts using DNA-containing liposomes. In someembodiments, marker genes are used in order to indicate whether or notthe gene of interest is present in the host cell. In some embodiments,the BBPI fusion polynucleotide sequence contained in the vector of theinvention encodes for a BBPI fusion protein having SEQ ID NO:195. Inaddition to these methods, in other embodiments, host cells are directlytransformed. In “direct transformation,” an intermediate cell is notused to amplify, or otherwise process, the modified polynucleotide priorto introduction into the host (i.e., Bacillus) cell. Introduction of themodified polynucleotide into the host cell includes those physical andchemical methods known in the art to introduce modified polynucleotideinto a host cell without insertion into a plasmid or vector. Suchmethods include but are not limited to the use of competent cells, aswell as the use of “artificial means” such as calcium chlorideprecipitation, electroporation, etc. to introduce DNA into cells. Thus,the present invention finds use with naked DNA, liposomes and the like.

Examples of suitable bacterial host organisms are Gram positive species,including, but not limited to members of the Bacillus species, whichBacillaceae, (e.g., B. subtilis, B. licheniformis, B. lentus, B. brevis,B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.clausii, B. halodurans, B. coagulans, B. circulans, B. lautus, B.megaterium and B. thuringiensis), Streptomyces species (e.g., S. murinusand S. lividans) lactic acid bacteria (e.g., Lactococcus spp. such asLactococcus lactis; Lactobacillus spp. including Lactobacillus reuteri;Leuconostoc spp.; Pediococcus spp.; and Streptococcus spp.Alternatively, strains of Gram-negative species belonging toEnterobacteriaceae (e.g., E. coli) or members of the Pseudomonadaceaefind use in the present invention.

In some embodiments, a suitable yeast host organism is selected fromvarious biotechnologically useful yeasts species, including but notlimited to Pichia sp., Hansenula sp or Kluyveromyces, Yarrowinia,Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyce(e.g., S. pombe). In some embodiments, strains of the methylotrophicyeast species Pichia pastoris are used as the host organism, while inother embodiments, the host organism is a Hansenula species. Suitablehost organisms among filamentous fungi include species of Aspergillus(e.g., A. niger, A. oryzae, A. tubigensis, A. awamori and Aspergillusnidulans). Alternatively, strains of Fusarium species (e.g. F.oxysporum) and Rhizomucor (e.g., Rhizomucor miehei) find used as thehost organism. Additional suitable strains include, but are not limitedto Thermomyces and Mucor species.

Accessory proteins such as thiol-disulfide oxidoreductases or chaperonesfind use in some embodiments, as they may be beneficial to help fold thesecretory protein into its active conformation. Thiol-disulfideoxidoreductases and protein disulfide isomerases catalyze the formationof the correct disulfide bonds in the protein. Overexpression of thebdbDC operon in B. subtilis has been shown to be beneficial for theproduction of a protein with disulfide bonds (See e.g., Meima et al., J.Biol. Chem., 277:6994-7001, [2002]). Chaperones help the secretoryprotein to fold by binding to exposed hydrophobic regions in theunfolded states and preventing unfavourable interactions andprolyl-peptidyl cis-trans isomerases assist in formation of the properconformation of the peptide chain adjacent to proline residues.

In some embodiments of the present invention, the host cells aretransformed with an expression vector encoding at least onethiol-disulfide oxidoreductase or chaperone. It is not intended that thepresent invention be limited to any particular thiol-disulfideoxidoreductase or chaperone, as any suitable thiol-disulfideoxidoreductase or chaperone known to those skilled in the art will finduse in the present invention.

In some embodiments of the present invention, and as described furtherbelow, the fraction of properly folded secretory protein is increased bythe addition of chemicals to the growth medium that reduce/oxidizedisulfide bonds, and/or alter the general redox potential, and/orchemicals that alter solvent properties thus affecting proteinconformation and aggregation. In particularly preferred embodiments, areagent that reduces disulfide bonds, such as 2-mercaptoethanol (βME),is preferred to increase the fraction of correctly folded protein.However, in other embodiments and depending on the medium used, otherdisulfide reducing or oxidizing agents (e.g., DTT, TCEP, reduced andoxidized glutathione, cysteine, cystine, cysteamine, thioglycolate, S₂O₃²⁻, S₂O₄ ²⁻, S₂O₅ ²⁻, SO₃ ²⁻, S₂O₇ ²⁻, Cu+, etc.), either used alone orin combination, find use in the present invention. It is contemplatedthat other adjuvants that alter solvent properties, (e.g., urea, DMSO,TWEEN®-80, etc.), either added to the growth medium alone or preferablyin combination with disulfide reducing/oxidizing agents, such as □ME,will also increase the fraction of correctly folded secretory proteinand find use in various embodiments of the present invention. In somepreferred embodiments, the BME is used at concentrations ranging from0.5 to 4 mM, while in other embodiments, the concentrations range from0.1 mM to 10 mM. Indeed, those of skill in the art know how to selectthe best growth medium and growth conditions to optimize the effects ofthe added thiol reducing/oxidizing agents and/or other adjuvants, aswell as the concentration of thio reducing/oxidizing agents and/or otheradjuvants to use. It is not intended that the present invention belimited to any particular disulfide reducing/oxidizing agent oradjuvant, as any suitable reagents known to those skilled in the artfind use in the present invention.

5.7.1 Fermentation Parameters

The present invention relies on fermentation procedures for culturingbacterial species. Fermentation procedures for production ofheterologous proteins by bacterial species are well known in the art.Culturing is accomplished in a growth medium comprising an aqueousmineral salts medium, organic growth factors, the carbon and energysource material, molecular oxygen (for aerobic and facultativebacteria), and, of course, a starting inoculum of one or more particularmicroorganism species to be employed.

In addition to the carbon and energy source, oxygen, assimilablenitrogen, and an inoculum of the microorganism, it is necessary tosupply suitable amounts in proper proportions of mineral nutrients toassure proper microorganism growth, maximize the assimilation of thecarbon and energy source by the cells in the microbial conversionprocess, and achieve maximum cellular yields with maximum cell densityin the fermentation medium.

Various culture media find use in the present invention, as known tothose of skill in the art. However, standard bacterial culture mediafind use in the present invention. In some preferred media formulations,the media include, in addition to nitrogen, suitable amounts ofphosphorus, magnesium, calcium, potassium, sulfur, and sodium, insuitable soluble assimilable ionic and combined forms, and also presentpreferably should be certain trace elements such as copper, manganese,molybdenum, zinc, iron, boron, and iodine, and others, again in suitablesoluble assimilable form, all as known in the art.

In some embodiments, the fermentation reaction involves an aerobicprocess in which the molecular oxygen needed is supplied by a molecularoxygen-containing gas such as air, oxygen-enriched air, or evensubstantially pure molecular oxygen, provided to maintain the contentsof the fermentation vessel with a suitable oxygen partial pressureeffective in assisting the microorganism species to grow in a thrivingfashion. In effect, by using an oxygenated hydrocarbon substrate, theoxygen requirement for growth of the microorganism is reduced.Nevertheless, molecular oxygen must be supplied for growth of aerobicand to a lesser extent, facultative organisms.

Although the aeration rate can vary over a considerable range, aerationgenerally is conducted at a rate which is in the range of about 0.5 to10, preferably about 0.5 to 7, volumes (at the pressure employed and at25° C.) of oxygen-containing gas per liquid volume in the fermentor perminute. This amount is based on air of normal oxygen content beingsupplied to the reactor, and in terms of pure oxygen the respectiveranges would be about 0.1 to 1.7, or preferably about 0.1 to 1.3,volumes (at the pressure employed and at 25° C.) of oxygen per liquidvolume in the fermentor per minute.

The pressure employed for the microbial conversion process can rangewidely. Pressures generally are within the range of about 0 to 50 psig,presently preferably about 0 to 30 psig, more preferably at leastslightly over atmospheric pressure, as a balance of equipment andoperating cost versus oxygen solubility achieved. Greater thanatmospheric pressures are advantageous in that such pressures do tend toincrease a dissolved oxygen concentration in the aqueous ferment, whichin turn can help increase cellular growth rates. At the same time, thisis balanced by the fact that high atmospheric pressures do increaseequipment and operating costs.

The fermentation temperature can vary somewhat, but for most bacterialspecies used in the present invention, the temperature generally will bewithin the range of about 20° C. to 40° C., generally preferably in therange of about 28° C. to 37° C., depending on the strain ofmicroorganism chosen, as known to those skilled in the art.

The microorganisms also require a source of assimilable nitrogen. Thesource of assimilable nitrogen can be any nitrogen-containing compoundor compounds capable of releasing nitrogen in a form suitable formetabolic utilization by the microorganism. While a variety of organicnitrogen source compounds, such as protein hydrolysates, can beemployed, usually cheap nitrogen-containing compounds such as ammonia,ammonium hydroxide, urea, and various ammonium salts such as ammoniumphosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride,or various other ammonium compounds can be utilized. Ammonia gas itselfis convenient for large scale operations, and can be employed bybubbling through the aqueous ferment (fermentation medium) in suitableamounts. At the same time, such ammonia can also be employed to assistin pH control.

The pH range in the aqueous microbial ferment (fermentation admixture)should be in the exemplary range of about 2.0 to 8.0. However, pH rangeoptima for certain microorganisms are dependent on the media employed tosome extent, as well as the particular microorganism, and thus changesomewhat with change in media as known to those skilled in the art.

While the average retention time of the fermentation admixture in thefermentor can vary considerably, depending in part on the fermentationtemperature and culture employed, as known in the art.

In some embodiments, the fermentation is preferably conducted in such amanner that the carbon-containing substrate can be controlled as alimiting factor, thereby providing good conversion of thecarbon-containing substrate to cells and avoiding contamination of thecells with a substantial amount of unconverted substrate. The latter isnot a problem with water-soluble substrates, since any remaining tracesare readily removed. It may be a problem, however, in the case ofnon-water-soluble substrates, and require added product-treatment stepssuch as suitable washing steps. The time needed to reach this limitingsubstrate level is not critical and may vary with the particularmicroorganism and fermentation process being conducted. However, it iswell known in the art how to determine the carbon source concentrationin the fermentation medium and whether or not the desired level ofcarbon source has been achieved.

Although in some embodiments, the fermentation is conducted as a batchor continuous operation, fed batch operation is generally preferred forease of control, production of uniform quantities of products, and mosteconomical uses of all equipment.

If desired, part or all of the carbon and energy source material and/orpart of the assimilable nitrogen source such as ammonia can be added tothe aqueous mineral medium prior to feeding the aqueous mineral mediuminto the fermentor. Indeed, each of the streams introduced into thereactor preferably is controlled at a predetermined rate, or in responseto a need determinable by monitoring such as concentration of the carbonand energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide inthe off-gases from the fermentor, cell density measurable by lighttransmittancy, or the like. The feed rates of the various materials canbe varied so as to obtain as rapid a cell growth rate as possible,consistent with efficient utilization of the carbon and energy source,to obtain as high a yield of microorganism cells relative to substratecharge as possible, but more importantly to obtain the highestproduction of the desired protein per unit volume.

In either a batch, or the preferred fed batch operation, all equipment,reactor, or fermentation means, vessel or container, piping, attendantcirculating or cooling devices, and the like, are initially sterilized,usually by employing steam such as at about 121° C. for at least about15 minutes. The sterilized reactor then is inoculated with a culture ofthe selected microorganism in the presence of all the requirednutrients, including oxygen, and the carbon-containing substrate. Thetype of fermentor employed is not critical, though in some embodiments,the 15L Biolafitte (Saint-Germain-en-Laye, France) is preferred.

5.8 Protein Separation

In some embodiments, host cells transformed with polynucleotidesequences encoding modified proteases are cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture. The protein produced by a recombinant host cell comprisinga fusion BBPI of the present invention is secreted into the culturemedia. In some embodiments, the secreted fusion BBPI is recovered.

In some embodiments, the present invention provides methods ofseparating a desired protein from its fusion analog. It is contemplatedthat the methods described herein will find use in the separation of themodified variant BBPI from the fusion analog.

The collection and purification of the desired fusion BBPI protein fromthe fermentation broth can also be achieved using procedures known tothose of skill in the art. The fermentation broth will generally containcellular debris, including cells, various suspended solids and otherbiomass contaminants, as well as the desired protein product, which arepreferably removed from the fermentation broth by means known in theart. Suitable processes for such removal include conventionalsolid-liquid separation techniques (e.g., centrifugation, filtration,dialysis, microfiltration, rotary vacuum filtration, or other knownprocesses), to produce a cell-free filtrate. In some embodiments, it ispreferable to further concentrate the fermentation broth or thecell-free filtrate prior to the purification and/or crystallizationprocess using techniques such as ultrafiltration, evaporation and/orprecipitation.

Precipitating the proteinaceous components of the supernatant orfiltrate may be accomplished by means of a salt (e.g., ammonium sulfate)or low pH (typically less than 3), followed by purification by a varietyof chromatographic procedures (e.g., ion exchange chromatography,affinity chromatography, hydrophobic interaction chromatography,hydrophobic charge induction chromatography etc.) or similar artrecognized procedures. It is not intended that the present invention belimited to any particular separation method, as it is contemplated thatany method will find use in the present invention.

In certain preferred embodiments, when the expressed desired polypeptideis secreted from the bacterial cells, the polypeptide is purified fromthe growth media. In preferred embodiments, the expression host cellsare removed from the media before purification of the polypeptide (e.g.by centrifugation).

When the expressed recombinant desired polypeptide is not secreted fromthe host cell, the host cell is preferably disrupted and the polypeptidereleased into an aqueous “extract” which is the first stage ofpurification. Preferably, the expression host cells are collected fromthe media before the cell disruption (e.g. by centrifugation). The celldisruption may be performed by using any suitable means known in theart, such as by lysozyme or beta-glucanase digestion or by forcing thecells through high pressure (See e.g., Scobes, Protein Purification,Second edition, Springer-Verlag)

In some embodiments, other recombinant constructions include theaddition of purification facilitating domains to the nucleotide sequenceencoding a modified variant BBPI polypeptide domain which facilitatespurification of the soluble proteins (Kroll D J et al (1993) DNA CellBiol 12:441-53). In some embodiments, the addition of six histidineresidues (i.e., a “His Tag”) to the C-terminus of the modified variantBBPI is used as an aid in the purification of the desired protein andits fusion analog. Use of the His tags as a purification aid is wellknown in the art (See e.g., Hengen, TIBS 20:285-286 [1995]). The 6×his-tagged proteins are easily purified using Immobilized Metal ionAffinity Chromatography (IMAC), as known to those skilled in the art.Other purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals (Porath J (1992) Protein Expr Purif3:263-281), protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego Calif.) between the purification domain and the heterologousprotein also find use to facilitate purification.

Thus, any method suitable for recovering the fusion BBPIs of the presentinvention finds use in the present invention. Indeed, it is not intendedthat the present invention be limited to any particular purificationmethod.

For some applications, it is of great importance that the proteaseinhibitors produced using the present invention be highly pure (e.g.,having a purity of more than 99%). This is particularly true wheneverthe desired protein is to be used as a therapeutic, but is alsonecessary for other applications. The methods described herein provide away of producing substantially pure desired proteins. The desiredproteins described herein are useful in pharmaceutical and personal carecompositions. However, it is contemplated that proteins of varyingpurity levels will be produced using the methods of the presentinvention and it is not intended that the proteins produced using thepresent invention be limited to any particular level of purity.

5.8.1 Activation of BBPI During Purification: Protease InhibitoryActivity

In some embodiments of the present invention, after growth during thepurification process, the activity of the protein i.e. the proteaseinhibitory activity, is increased by the addition of chemicals thatreduce/oxidize disulfide bonds and/or alter the general redox potential,and/or chemicals that alter solvent properties thus affecting proteinconformation and aggregation. In some particularly preferredembodiments, addition of a reagent that reduces disulfide bonds, such as2-mercaptoethanol, is used to increase activity of the protein. However,as those skilled in the art appreciate, depending purity and buffercomposition, other disulfide reducing or oxidizing agents (e.g., DTT,TCEP, reduced and oxidized glutathione, cysteine, cystine, cysteamine,thioglycolate, S₂O₃ ²⁻, S₂O₄ ²⁻, S₂O₅ ²⁻, SO₃ ²⁻, S₂O₇ ²⁻, Cu+, proteindisulfide isomerases, protein thiol-disulfide oxidoreductases, etc.),either used alone or in combination, find use in the present invention.Other adjuvants, which alter solvent properties, (e.g. ethanolamine,DMSO, TWEEN®-80, arginine, urea, etc.), are either added alone orpreferably in combination with disulfide reducing/oxidizing agents, suchas βME, during the purification process also find use in the presentinvention by increasing the activity of the protein. In certainpreferred embodiments, partially purified protein is diluted in buffer(in some particularly preferred embodiments, a zwitterionic buffer withTWEEN®-80 at basic pH) and activated with βME and a disulfide oxidizingagent (in alternative preferred embodiments, oxidized glutathione orsodium sulfite).

In addition, it is contemplated that conditions will be screened inorder to determine the optimal activation of the BBPI protein, ifdesired. For example, various βME concentrations (0.1-10 mM), oxidizingagent concentrations (0 to 1/20 to 20 times the βME concentration) pH(7.5-9.5), temperatures (15-40° C.), dilutions (1-20 fold), incubationtimes (12-72 h), aeration (incubations under inert gas to vigorousmixing under oxygen containing gases), buffer types (Tris, CHES, CAPS,Tricine, TAPS, other zwitterionic buffers, etc.), buffer concentrations(0.1-1 M), and the addition of various adjuvants known to alter solventproperties thereby affecting protein conformation and aggregation (e.g.,ethanolamine, DMSO, TWEEN®-80, arginine, urea, etc.) are tested in orderto determine the optimum conditions for the expression system used. Itis not intended that the present invention be limited to any particulardisulfide reducing/oxidizing agent, dilution, temperature, pH, buffertype or composition, or adjuvant, as any suitable reagents known tothose skilled in the art find use in the present invention.

Optimal activation of BBPIs by thiol reducing agents and/or oxidizingagents is monitored by measuring an increase in the protease inhibitoryactivity of the unmodified variant BBPI. Trypsin inhibitory activity isthe protease inhibitory that is assayed in variant BBPIs in which thechymotrypsin loop has been replaced by a variant peptide, whilechymotrypsin inhibitory activity is the protease inhibitory that isassayed in variant BBPIs in which the trypsin loop has been replaced bya variant peptide. In some embodiments, the effect of at least one aminoacid substitution on the trypsin inhibitory activity of a modified BBPIis assayed and compared to the trypsin inhibitory activity of theunmodified precursor BBPI.

In some embodiments, modified variant BBPIs comprising at least oneamino acid substitution have greater trypsin inhibitory activity thanthat of the unmodified precursor BBPI. In some embodiments, the singleamino acid substitution that generates a modified variant BBPI having agreater TIA than the unmodified precursor is chosen from equivalent to aposition chosen from positions equivalent to positions 1, 4, 5, 11, 13,18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187 resultin the following substituted amino acids. In one embodiment, thesubstituted amino acid at the amino acid position equivalent to position1 of SEQ ID NO:187 is chosen from A and C. In another embodiment, thesubstituted amino acid at the amino acid position equivalent to position4 of SEQ ID NO:187 is V. In another embodiment, the substituted aminoacid at the amino acid position equivalent to position 5 of SEQ IDNO:187 is chosen from P, and A. In another embodiment, the substitutedamino acid at the amino acid position equivalent to position 11 of SEQID NO:187 is G. In another embodiment, the substituted amino acid at theamino acid position equivalent to position 13 of SEQ ID NO:187 is chosenfrom Y, I, F, M, L, V, K, and R. In another embodiment, the substitutedamino acid at the amino acid position equivalent to position 18 of SEQID NO:187 include 1, V and L. In another embodiment, the substitutedamino acid at the amino acid position equivalent to position 25 of SEQID NO:187 is chosen from K, N, W, I, A and R. In another embodiment, thesubstituted amino acid at the amino acid position equivalent to position27 of SEQ ID NO:187 include R, K, V, A, and Q. In another embodiment,the substituted amino acid at the amino acid position equivalent toposition 29 of SEQ ID NO:187 is chosen from R, K, and P. In anotherembodiment, the substituted amino acid at the amino acid positionequivalent to position 31 of SEQ ID NO:187 is chosen from Q, H, E, A, R,W, K and T. In another embodiment, the substituted amino acid at theamino acid position equivalent to position 38 of SEQ ID NO:187 is chosenfrom N, K and R. In another embodiment, the substituted amino acid atthe amino acid position equivalent to position 40 of SEQ ID NO:187 ischosen from H, K, Q, R, and Y. In another embodiment, the substitutedamino acid at the amino acid position equivalent to position 50 of SEQID NO:187 is chosen from R, Q, K, T, V, M, and S. In another embodiment,the substituted amino acid at the amino acid position equivalent toposition 52 of SEQ ID NO:187 is chosen from K, T, R, Q, L, H, A, M, Sand E. In another embodiment, the substituted amino acid at the aminoacid position equivalent to position 55 of SEQ ID NO:187 is M. Inanother embodiment, the substituted amino acid at the amino acidposition equivalent to position 65 of SEQ ID NO:187 is chosen from E, Q,and D.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of two amino acid substitutions at amino acids at positionsequivalent to positions 50 and 52 of SEQ ID NO:187. In some embodiments,the combination of two amino acid substitutions is 50T-52A. Theinvention provides for any one of the variant BBPI scaffolds describedin Section 5.3 and further comprising the combination of the two aminoacid substitutions 50T-52A, as described in section 5.4. In oneembodiment, the chymotrypsin loop of the variant scaffold is a VEGFvariant peptide e.g. SEQ ID NO:9, and the variant scaffold is alteredfurther to comprise a combination of three amino acid substitutions atpositions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 togenerate a modified variant BBPI scaffold. In one embodiment, themodified variant BBPI comprising a combination of two amino acidsubstitutions is the modified variant BBIt-AV-F50T-V52A of SEQ IDNO:595.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of three amino acid substitutions at amino acids atpositions equivalent to positions 13, 50 and 52 of SEQ ID NO:187. Insome embodiments, the combination of three amino acid substitutions ischosen from a combination of substitutions at positions 25-50-52,29-50-52, 40-50-52, and 13-50-52. In some embodiments, the combinationof three amino acid substitutions is chosen from 25L-50T-52A,29P-50T-52A, 40K-50T-52A and 13I-50T-52A. The invention provides for anyone of the variant BBPI scaffolds described in 5.3 and furthercomprising the combination of the three amino acid substitutions chosenfrom 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A, as describedin section 5.4. In one embodiment, the chymotrypsin loop of the variantscaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variantscaffold is altered further to comprise a combination of three aminoacid substitutions at positions equivalent to positions 13, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of threeamino acid substitutions is chosen from the modified variantBBIt-AV-S25L-F50T-V52A of SEQ ID NO: 603, the modified variantBBIt-AV-L29P-F50T-V52A of SEQ ID NO:607, and the modified variantBBIt-AV-A40K-F50T-V52A of SEQ ID NO:609.

In some embodiments, the modified variant BBPI that have greater TIAthan the corresponding precursor unmodified BBPIs each comprise acombination of four amino acid substitutions at amino acids at positionsequivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of four amino acid substitutions is chosenfrom a combination of substitutions at positions 13-25-50-52,13-29-50-52, 25-29-50-52, 13-40-50-52, 25-40-50-52, and 29-40-50-52. Insome embodiments, the combination of four amino acid substitutions ischosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A,13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A. The inventionprovides for any one of the variant BBPI scaffolds described in Section5.3 and further comprising the combination of the four amino acidsubstitutions chosen from 13I-25L-50T-52A, 13I-29P-50T-52A,25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A,as described in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide chosen from SEQ ID NO:9and 460, and the variant scaffold is altered further to comprise acombination of four amino acid substitutions at positions equivalent topositions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modifiedvariant BBPI scaffold. In one embodiment, the modified variant BBPIcomprising a combination of four amino acid substitutions is chosen fromthe modified variant BBIt-AV-A13I-S25L-F50T-V52A of SEQ ID NO:596, themodified variant BBIt-AV-A13I-L29P-F50T-V52A of SEQ ID NO:600, themodified variant BBIt-AV-A13I-A40K-F50T-V52A of SEQ ID NO:602, themodified variant BBIt-AV-S25L-L29P-F50T-V52A of SEQ ID NO:604, themodified variant BBIt-AV-S25L-A40K-F50T-V52A of SEQ ID NO:606, themodified variant BBIt-AV-L29P-A40K-F50T-V52A of SEQ ID NO:608, and themodified variant BBIt-VEGKD-A13I-S25K-L29P-V52K of SEQ ID NO:643. Inanother embodiment, the chymotrypsin loop of the variant scaffold is anFGF5 variant peptide chosen from SEQ ID NOS:433 and 434, and the variantscaffold is altered further to comprise a combination of four amino acidsubstitutions at positions equivalent to positions 13, 29, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of fouramino acid substitutions is chosen from the modified variantBBIt-MM007-Q-A13I-L29P-F50T-V52A of SEQ ID NO:432, and the modifiedvariant BBIt-FGFps2-Q-A13I-L29P-F50T-V52A of SEQ ID NO:434. In anotherembodiment, the chymotrypsin loop of the variant scaffold is a TGFβvariant peptide chosen from SEQ ID NOS:436, 437 and 438, and the variantscaffold is altered further to comprise a combination of four amino acidsubstitutions at positions equivalent to positions 13, 29, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of fouramino acid substitutions is chosen from the modified variantBBIt-PEN3-Q-A13I-L29P-F50T-V52A of SEQ ID NO:443, the modified variantBBIt-MM021W-Q-A13I-L29P-F50T-V52A of SEQ ID NO:445, and the modifiedvariant BBIt-WTQ-Q-A13I-L29P-F50T-V52A of SEQ ID NO:447.

In some embodiments, the modified variant BBPI that have greater TIAthan the corresponding precursor unmodified BBPIs each comprise acombination of five amino acid substitutions at amino acids at positionsequivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of five amino acid substitutions is chosenfrom a combination of substitutions at positions 13-25-29-50-52,13-29-40-50-52, 13-25-40-50-52, 25-29-40-50-52, 13-29-40-50-52,13-29-40-50-52, 13-29-40-50-52 and 13-29-40-50-52. In some embodiments,the combination of five amino acid substitutions is chosen from13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A,25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A,13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T. The invention provides forany one of the variant BBPI scaffolds described in Section 5.3 andfurther comprising the combination of the five amino acid substitutionschosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A,13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A,13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T, asdescribed in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and thevariant scaffold is altered further to comprise a combination of fiveamino acid substitutions at positions equivalent to positions 13, 29,40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPIscaffold. In one embodiment, the modified variant BBPI comprising acombination of five amino acid substitutions is chosen from the modifiedvariant BBIt-AV-A13I-S25L-L29P-F50T-V52A of SEQ ID NO:597, the modifiedvariant BBIt-AV-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:599, the modifiedvariant BBIt-AV-A13I-S25L-A40K-F50T-V52A of SEQ ID NO:601, the modifiedvariant BBIt-AV-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:605, the modifiedvariant BBIt-AV-A13L-L29P-A40K-F50T-V52A of SEQ ID NO:615, the modifiedvariant BBIt-AV-A13I-L29K-A40K-F50T-V52A of SEQ ID NO:620, the modifiedvariant BBIt-AV-A13I-L29P-A40K-F50K-V52A of SEQ ID NO:624, and themodified variant BBIt-AV-A13I-L29P-A40K-F50T-V52T of SEQ ID NO:625.

In some embodiments, the modified variant BBPI that have greater TIAthan the corresponding precursor unmodified BBPIs each comprise acombination of six amino acid substitutions at amino acids at positionsequivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. Insome embodiments, the combination of six amino acid substitutions ischosen from a combination of substitutions at positions13-25-29-40-50-52, 1-13-29-40-50-52, 4-13-29-40-50-52, 5-13-29-40-50-52,11-13-29-40-50-52, 13-25-29-40-50-52, 13-27-29-40-50-52,13-29-31-40-50-52, 13-29-31-40-50-52, 13-29-38-40-50-52, and13-29-38-40-50-52. In some embodiments, the combination of six aminoacid substitutions is chosen from 13I-25L-29P-40K-50T-52A,1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A,11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A,13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A,13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and13I-29P-38N-40K-50T-52A. The invention provides for any one of thevariant BBPI scaffolds described in Section 5.3 and further comprisingthe combination of the six amino acid substitutions chosen from13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A,5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A,13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A,13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A,13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A, as described insection 5.4. In one embodiment, the chymotrypsin loop of the variantscaffold is a VEGF variant peptide of SEQ ID NO:9, and the variantscaffold is altered further to comprise a combination of six amino acidsubstitutions at positions equivalent to positions 13, 25, 29, 40, 50and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. Inone embodiment, the modified variant BBPI comprising a combination ofsix amino acid substitutions is chosen from the modified variantBBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:598, the modifiedvariant BBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:611, themodified variant BBIt-AV-S4V-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:612,the modified variant BBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A of SEQ IDNO:613, the modified variant BBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A ofSEQ ID NO:614, the modified variantBBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A- of SEQ ID NO:616, the modifiedvariant BBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A of SEQ ID NO:619, themodified variant BBIt-AV-A13I-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:621,the modified variant BBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A of SEQ IDNO:622, the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A ofSEQ ID NO:623, and the modified variantBBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:626.

In some embodiments, the modified variant BBPI that have greater TIAthan the corresponding precursor unmodified BBPIs each comprise acombination of seven amino acid substitutions at amino acids atpositions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQID NO:187. In some embodiments, the combination of seven amino acidsubstitutions is chosen from a combination of substitutions at positions13-25-29-31-40-50-52, 13-25-29-31-40-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, and 13-25-27-29-31-50-52. In some embodiments, thecombination of seven amino acid substitutions is chosen from13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and13I-25R-27A-29P-31A-50K-52T. The invention provides for any one of thevariant BBPI scaffolds described in Section 5.3 and further comprisingthe combination of the six amino acid substitutions chosen from13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and13I-25R-27A-29P-31A-50K-52T, as described in section 5.4. In oneembodiment, the chymotrypsin loop of the variant scaffold is a VEGFvariant peptide of SEQ ID NO:9, and the variant scaffold is alteredfurther to comprise a combination of seven amino acid substitutions atpositions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of sevenamino acid substitutions is chosen from the modified variantBBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:617, themodified variant BBIt-AV-A13L-S25R-L29P-S31R-A40K-F50T-V52A of SEQ IDNO:618, the modified variant ofBBIt-VEGF-V1-A13I-S25R-M27A-L29P-S31A-F50K-V52T SEQ ID NO:491, themodified variant BBIt-VEGF-V2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:632, the modified variantBBIt-VEGF-V3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:633, themodified variant BBIt-VEGF-V4-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:634, the modified variantBBIt-VEGF-V5-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:635, themodified variant BBIt-VEGF-V6-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:636, the modified variantBBIt-TNFα-T1-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:637, themodified variant BBIt-TNFα-T2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:638, and the modified variantBBIt-TNFα-T3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:639.

In some embodiments, the modified variant BBPI that have greater TIAthan the corresponding precursor unmodified BBPIs each comprise acombination of eight amino acid substitutions at amino acids atpositions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 ofSEQ ID NO:187. In some embodiments, the combination of eight amino acidsubstitutions is chosen from a combination of substitutions at positions13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52,13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, and13-25-27-29-31-40-50-52. In some embodiments, the combination of eightamino acid substitutions is chosen from combinations13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q,13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and13I-25K-27Q-29P-31E-40H-50R-52Q. The invention provides for any one ofthe variant BBPI scaffolds described in Section 5.3 and furthercomprising the combination of the eight amino acid substitutions chosenfrom combinations 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K,13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q, asdescribed in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and thevariant scaffold is altered further to comprise a combination of sevenamino acid substitutions at positions equivalent to positions 13, 25,27, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modifiedvariant BBPI scaffold. In one embodiment, the modified variant BBPIcomprising a combination of eight amino acid substitutions is chosenfrom the modified variantBBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T of SEQ ID NO:627, themodified variant of BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q ofSEQ ID NO:628, the modified variantBBIt-AV-A13I-S25K-M27R-L29E-S31A-A40H-F50R-V52K of SEQ ID NO:629, themodified variant BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L of SEQID NO:630, and the modified variantBBIt-AV-A13I-S25K-M27Q-L29P-S31E-A40H-F50R-V52Q of SEQ ID NO:631.

In some embodiments, the modified variant BBPI that have greater TIAthan the corresponding precursor unmodified BBPIs each further comprisea peptide insert that is positioned at the N-terminus of the modifiedvariant BBPI. In some embodiments, the peptide insert comprises asequence of between 1 and 15 amino acids. In other embodiments, thepeptide insert comprises a sequence between 5 and 10 amino acids. Insome embodiments, the peptide insert comprises the peptide of SEQ IDNO:389 (DDEPSKPCCDPDP; SEQ ID NO:389). Examples of modified variantBBPIs that the peptide insert of SEQ ID NO:389 are the modified variant4D13BBIt-AV of SEQ ID NO:390(DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE;SEQ ID NO:390), and the modified variantBBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413.

One measure of enhancement in trypsin and/or chymotrypsin inhibitoryactivity can be determined as the increase in an enzymatic activityratio i.e. BCE:BBPI, in the modified variant BBPI when compared to thatof the unmodified variant BBPI. The BCE:BBPI enzymatic activity ratio isthe ratio of BBPI protease inhibitory activity e.g. trypsin inhibitoryactivity, to the BCE i.e. cellulase enzymatic activity. The ratio of theunmodified variant BBPI is assigned a value of 1. A ratio equal orgreater than 1 for a modified variant BBPI indicates that the modifiedvariant BBPI has a greater protease inhibitory activity e.g. trypsininhibitory activity than that of the unmodified precursor BBPI. In someembodiments, the activity ratio of the modified variant BBPI is at least1, at least about 1.05, about at least about 1.1, at least about 1.2, atleast about 1.3, at least about 1.4, at least about 1.5, at least about1.6, at least about 1.7, at least about 1.8. at least about 1.9, and atleast about 2. In other embodiments, the activity ratio is at leastabout 2.1, at least about 2.2, at least about 2.3, at least about 2.4,at least about 2.5, at least about 2.6, at least about 2.7, at leastabout 2.8, at least about 2.9 and at least about 3. In yet otherembodiments, the activity ratio is at least about 3.5, at least about4.0, and at least about 5. Thus, in some embodiments, the proteaseinhibitory activity e.g. protease inhibitory activity, of the modifiedvariant BBPI greater than that of the corresponding unmodified precursorBBPI by at least about 0.5%, about 1.0%, about 1.5%, about 2.0%, about2.5%, about 3.0%, about 4.0%, about 5.0%, about 8.0%, about 10%, about15%, about 20%, about 25%, about 30%, about 40%, about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 100% or more. In other embodiments, the proteaseinhibitory activity e.g. protease inhibitory activity, of the modifiedvariant BBPI greater than that of the corresponding unmodified precursorBBPI by at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, and up to at least about 200%.

In other embodiments, modified variant BBPIs comprising a combination ofamino acid substitutions have greater trypsin inhibitory activity thanthat of the unmodified precursor BBPI. In some embodiments, the at leasttwo, at least three, at least four, at least five, at least six, atleast seven, and at least eight amino acid substitutions generatemodified variant BBPIs that have greater TIA than the unmodifiedprecursor variant BBPI. All modified variant BBPIs comprising acombination of amino acid substitutions as described in section 5.4 havegreater trypsin inhibitory activity than the corresponding unmodifiedprecursor BBPIs.

5.8.2 BBPI Production Yield

In some embodiments, the amino acid substitutions made in the precursorvariant BBPI are assessed for the ability of the resulting modifiedvariant BBPI to have a greater production yield that is greater thanthat at which the unmodified precursor BBPI is produced i.e. themodified variant BBPI is produced at a level that is greater than thatat which the unmodified precursor BBPI is produced. In some embodiments,the invention provides for modified variant BBPIs comprising acombination of amino acid substitutions as described in section 5.4 thathave greater protease inhibitory activity e.g. trypsin inhibitoryactivity, and greater production yield than the corresponding unmodifiedprecursor BBPIs.

Modified variant BBPIs that have greater TIA and PY than thecorresponding precursor unmodified BBPIs each comprise at least two, atleast three, at least four, at least five, at least six, at least seven,and at least eight amino acid substitutions (see Examples 12, 13, 14 and15).

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of two amino acid substitutions at amino acids at positionsequivalent to positions 50 and 52 of SEQ ID NO:187. In some embodiments,the combination of two amino acid substitutions is 50T-52A. Theinvention provides for any one of the variant BBPI scaffolds describedin Section 5.3 and further comprising the combination of the two aminoacid substitutions 50T-52A, as described in section 5.4. In oneembodiment, the chymotrypsin loop of the variant scaffold is a VEGFvariant peptide e.g. SEQ ID NO:9, and the variant scaffold is alteredfurther to comprise a combination of three amino acid substitutions atpositions equivalent to positions 13, 50 and 52 of SEQ ID NO:187 togenerate a modified variant BBPI scaffold. In one embodiment, themodified variant BBPI comprising a combination of two amino acidsubstitutions is the modified variant BBIt-AV-F50T-V52A of SEQ ID NO:6595.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of three amino acid substitutions at amino acids atpositions equivalent to positions 13, 50 and 52 of SEQ ID NO:187. Insome embodiments, the combination of three amino acid substitutions ischosen from a combination of substitutions at positions 25-50-52,29-50-52, 40-50-52, and 13-50-52. In some embodiments, the combinationof three amino acid substitutions is chosen from 25L-50T-52A,29P-50T-52A, 40K-50T-52A and 13I-50T-52A. The invention provides for anyone of the variant BBPI scaffolds described in Section 5.3 and furthercomprising the combination of the three amino acid substitutions chosenfrom 25L-50T-52A, 29P-50T-52A, 40K-50T-52A and 13I-50T-52A, as describedin section 5.4. In one embodiment, the chymotrypsin loop of the variantscaffold is a VEGF variant peptide e.g. SEQ ID NO:9, and the variantscaffold is altered further to comprise a combination of three aminoacid substitutions at positions equivalent to positions 13, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of threeamino acid substitutions is chosen from the modified variantBBIt-AV-S25L-F50T-V52A of SEQ ID NO: 603, the modified variantBBIt-AV-L29P-F50T-V52A of SEQ ID NO:607, and the modified variantBBIt-AV-A40K-F50T-V52A of SEQ ID NO:609.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of four amino acid substitutions at amino acids at positionsequivalent to positions 13, 29, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of four amino acid substitutions is chosenfrom a combination of substitutions at positions 13-25-50-52,13-29-50-52, 25-29-50-52, 13-40-50-52, 25-40-50-52, and 29-40-50-52. Insome embodiments, the combination of four amino acid substitutions ischosen from 13I-25L-50T-52A, 13I-29P-50T-52A, 25L-29P-50T-52A,13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A. The inventionprovides for any one of the variant BBPI scaffolds described in Section5.3 and further comprising the combination of the four amino acidsubstitutions chosen from 13I-25L-50T-52A, 13I-29P-50T-52A,25L-29P-50T-52A, 13I-40K-50T-52A, 25L-40K-50T-52A, and 29P-40K-50T-52A,as described in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide chosen from SEQ ID NO:9and 460, and the variant scaffold is altered further to comprise acombination of four amino acid substitutions at positions equivalent topositions 13, 29, 50 and 52 of SEQ ID NO:187 to generate a modifiedvariant BBPI scaffold. In one embodiment, the modified variant BBPIcomprising a combination of four amino acid substitutions is chosen fromthe modified variant BBIt-AV-A13I-S25L-F50T-V52A of SEQ ID NO:596, themodified variant BBIt-AV-A13I-L29P-F50T-V52A of SEQ ID NO:600, themodified variant BBIt-AV-A13I-A40K-F50T-V52A of SEQ ID NO:602, themodified variant BBIt-AV-S25L-L29P-F50T-V52A of SEQ ID NO:604, themodified variant BBIt-AV-S25L-A40K-F50T-V52A of SEQ ID NO:606, themodified variant BBIt-AV-L29P-A40K-F50T-V52A of SEQ ID NO:608, and themodified variant BBIt-VEGKD-A13I-S25K-L29P-V52K of SEQ ID NO:643. Inanother embodiment, the chymotrypsin loop of the variant scaffold is anFGF5 variant peptide chosen from SEQ ID NOS:430 and 431, and the variantscaffold is altered further to comprise a combination of four amino acidsubstitutions at positions equivalent to positions 13, 29, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of fouramino acid substitutions is chosen from the modified variantBBIt-MM007-Q-A13I-L29P-F50T-V52A of SEQ ID NO:432, and the modifiedvariant BBIt-FGFps2-Q-A13I-L29P-F50T-V52A of SEQ ID NO:434. In anotherembodiment, the chymotrypsin loop of the variant scaffold is a TGFβvariant peptide chosen from SEQ ID NOS:436, 437 and 438, and the variantscaffold is altered further to comprise a combination of four amino acidsubstitutions at positions equivalent to positions 13, 29, 50 and 52 ofSEQ ID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of fouramino acid substitutions is chosen from the modified variantBBIt-PEN3-Q-A13I-L29P-F50T-V52A of SEQ ID NO:443, the modified variantBBIt-MM021W-Q-A13I-L29P-F50T-V52A of SEQ ID NO:445, and the modifiedvariant BBIt-WTQ-Q-A13I-L29P-F50T-V52A of SEQ ID NO:447.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of five amino acid substitutions at amino acids at positionsequivalent to positions 13, 29, 40, 50 and 52 of SEQ ID NO:187. In someembodiments, the combination of five amino acid substitutions is chosenfrom a combination of substitutions at positions 13-25-29-50-52,13-29-40-50-52, 13-25-40-50-52, 25-29-40-50-52, 13-29-40-50-52,13-29-40-50-52, 13-29-40-50-52 and 13-29-40-50-52. In some embodiments,the combination of five amino acid substitutions is chosen from13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A, 13I-25L-40K-50T-52A,25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A, 13I-29K-40K-50T-52A,13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T. The invention provides forany one of the variant BBPI scaffolds described in Section 5.3 andfurther comprising the combination of the five amino acid substitutionschosen from 13I-25L-29P-50T-52A, 13I-29P-40K-50T-52A,13I-25L-40K-50T-52A, 25L-29P-40K-50T-52A, 13L-29P-40K-50T-52A,13I-29K-40K-50T-52A, 13I-29P-40K-50K-52A and 13I-29P-40K-50T-52T, asdescribed in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and thevariant scaffold is altered further to comprise a combination of fiveamino acid substitutions at positions equivalent to positions 13, 29,40, 50 and 52 of SEQ ID NO:187 to generate a modified variant BBPIscaffold. In one embodiment, the modified variant BBPI comprising acombination of five amino acid substitutions is chosen from the modifiedvariant BBIt-AV-A13I-S25L-L29P-F50T-V52A of SEQ ID NO:597, the modifiedvariant BBIt-AV-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:599, the modifiedvariant BBIt-AV-A13I-S25L-A40K-F50T-V52A of SEQ ID NO:601, the modifiedvariant BBIt-AV-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:605, the modifiedvariant BBIt-AV-A13L-L29P-A40K-F50T-V52A of SEQ ID NO:615, the modifiedvariant BBIt-AV-A13I-L29K-A40K-F50T-V52A of SEQ ID NO:620, the modifiedvariant BBIt-AV-A13I-L29P-A40K-F50K-V52A of SEQ ID NO:624, and themodified variant BBIt-AV-A13I-L29P-A40K-F50T-V52T of SEQ ID NO:625.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of six amino acid substitutions at amino acids at positionsequivalent to positions 13, 25, 29, 40, 50 and 52 of SEQ ID NO:187. Insome embodiments, the combination of six amino acid substitutions ischosen from a combination of substitutions at positions13-25-29-40-50-52, 1-13-29-40-50-52, 4-13-29-40-50-52, 5-13-29-40-50-52,11-13-29-40-50-52, 13-25-29-40-50-52, 13-27-29-40-50-52,13-29-31-40-50-52, 13-29-31-40-50-52, 13-29-38-40-50-52, and13-29-38-40-50-52. In some embodiments, the combination of six aminoacid substitutions is chosen from 13I-25L-29P-40K-50T-52A,1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A,11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A,13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A,13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and13I-29P-38N-40K-50T-52A. The invention provides for any one of thevariant BBPI scaffolds described in Section 5.3 and further comprisingthe combination of the six amino acid substitutions chosen from13I-25L-29P-40K-50T-52A, 1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A,5P-13I-29P-40K-50T-52A, 11G-13I-29P-40K-50T-52A,13I-25R-29P-40K-50T-52A, 13I-27R-29P-40K-50T-52A,13I-29P-31A-40K-50T-52A, 13I-29P-31R-40K-50T-52A,13I-29P-38N-40K-50T-52A, and 13I-29P-38N-40K-50T-52A, as described insection 5.4. In one embodiment, the chymotrypsin loop of the variantscaffold is a VEGF variant peptide of SEQ ID NO:9, and the variantscaffold is altered further to comprise a combination of six amino acidsubstitutions at positions equivalent to positions 13, 25, 29, 40, 50and 52 of SEQ ID NO:187 to generate a modified variant BBPI scaffold. Inone embodiment, the modified variant BBPI comprising a combination ofsix amino acid substitutions is chosen from the modified variantBBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A of SEQ ID NO:598, the modifiedvariant BBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:611, themodified variant BBIt-AV-S4V-A13I-L29P-A40K-F50T-V52A of SEQ ID NO:612,the modified variant BBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A of SEQ IDNO:613, the modified variant BBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A ofSEQ ID NO:614, the modified variantBBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A- of SEQ ID NO:616, the modifiedvariant BBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A of SEQ ID NO:619, themodified variant BBIt-AV-A13I-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:621,the modified variant BBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A of SEQ IDNO:622, the modified variant BBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A ofSEQ ID NO:623, and the modified variantBBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A of SEQ ID NO:626.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of seven amino acid substitutions at amino acids atpositions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQID NO:187. In some embodiments, the combination of seven amino acidsubstitutions is chosen from a combination of substitutions at positions13-25-29-31-40-50-52, 13-25-29-31-40-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, 13-25-27-29-31-50-52, 13-25-27-29-31-50-52,13-25-27-29-31-50-52, and 13-25-27-29-31-50-52. In some embodiments, thecombination of seven amino acid substitutions is chosen from13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and13I-25R-27A-29P-31A-50K-52T. The invention provides for any one of thevariant BBPI scaffolds described in Section 5.3 and further comprisingthe combination of the six amino acid substitutions chosen from13L-25R-29P-31A-40K-50T-52A, 13L-25R-29P-31R-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-50K-52T, and13I-25R-27A-29P-31A-50K-52T, as described in section 5.4. In oneembodiment, the chymotrypsin loop of the variant scaffold is a VEGFvariant peptide of SEQ ID NO:9, and the variant scaffold is alteredfurther to comprise a combination of seven amino acid substitutions atpositions equivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQID NO:187 to generate a modified variant BBPI scaffold. In oneembodiment, the modified variant BBPI comprising a combination of sevenamino acid substitutions is chosen from the modified variantBBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A of SEQ ID NO:617, themodified variant BBIt-AV-A13I-S25R-L29P-S31R-A40K-F50T-V52A of SEQ IDNO:618, the modified variant ofBBIt-VEGF-V1-A13I-S25R-M27A-L29P-S31A-F50K-V52T SEQ ID NO:491, themodified variant BBIt-VEGF-V2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:632, the modified variantBBIt-VEGF-V3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:633, themodified variant BBIt-VEGF-V4-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:634, the modified variantBBIt-VEGF-V5-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:635, themodified variant BBIt-VEGF-V6-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:636, the modified variantBBIt-TNFα-T1-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:637, themodified variant BBIt-TNFα-T2-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQID NO:638, and the modified variantBBIt-TNFα-T3-A13I-S25R-M27A-L29P-S31A-F50K-V52T of SEQ ID NO:639.

In some embodiments, the modified variant BBPI that have greater TIA andPY than the corresponding precursor unmodified BBPIs each comprise acombination of eight amino acid substitutions at amino acids atpositions equivalent to positions 13, 25, 27, 29, 31, 40, 50 and 52 ofSEQ ID NO:187. In some embodiments, the combination of eight amino acidsubstitutions is chosen from a combination of substitutions at positions13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52,13-25-27-29-31-40-50-52, 13-25-27-29-31-40-50-52, and13-25-27-29-31-40-50-52. In some embodiments, the combination of eightamino acid substitutions is chosen from combinations13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q,13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L, and13I-25K-27Q-29P-31E-40H-50R-52Q. The invention provides for any one ofthe variant BBPI scaffolds described in Section 5.3 and furthercomprising the combination of the eight amino acid substitutions chosenfrom combinations 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27R-29E-31A-40H-50R-52K,13I-25K-27A-29R-31A-40H-50R-52L, and 13I-25K-27Q-29P-31E-40H-50R-52Q, asdescribed in section 5.4. In one embodiment, the chymotrypsin loop ofthe variant scaffold is a VEGF variant peptide of SEQ ID NO:9, and thevariant scaffold is altered further to comprise a combination of sevenamino acid substitutions at positions equivalent to positions 13, 25,27, 29, 31, 40, 50 and 52 of SEQ ID NO:187 to generate a modifiedvariant BBPI scaffold. In one embodiment, the modified variant BBPIcomprising a combination of eight amino acid substitutions is chosenfrom the modified variantBBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T of SEQ ID NO:627, themodified variant of BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q ofSEQ ID NO:628, the modified variantBBIt-AV-A13I-S25K-M27R-L29E-S31A-A40H-F50R-V52K of SEQ ID NO:629, themodified variant BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L of SEQID NO:630, and the modified variantBBIt-AV-A13I-S25K-M27Q-L29P-S31E-A40H-F50R-V52Q of SEQ ID NO:631.

In some embodiments, the modified variant BBPIs further comprise apeptide insert that is positioned at the N-terminus of the modifiedvariant BBPI. In some embodiments, the peptide insert comprises asequence of between 1 and 15 amino acids. In other embodiments, thepeptide insert comprises a sequence between 5 and 10 amino acids. Insome embodiments, the peptide insert comprises the peptide of SEQ IDNO:389. Examples of modified variant BBPIs that the peptide insert ofSEQ ID NO:389 are the modified variant 4D13BBIt-AV of SEQ ID NO:390, andthe modified variant BBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413.

The invention further provides for modified variant BBPIs that havegreater TIA and PY than the corresponding precursor unmodified BBPIseach comprise any one combination of the amino acid substitutionsdescribed above and that have greater protease inhibitory activity thanthe unmodified precursor variant BBPI. In some embodiments, modifiedvariant BBPIs which contain a variant peptide in place of thechymotrypsin loop of the corresponding precursor unmodified BBPI havegreater trypsin inhibitory activity (TIA) than that of the precursorunmodified BBPI scaffold. In other embodiments, modified variant BBPIswhich contain a variant peptide in place of the trypsin loop of thecorresponding precursor unmodified BBPI have greater chymotrypsininhibitory activity (TIA) than that of the precursor unmodified BBPIscaffold.

As shown in the Examples, substitutions of at least one amino acid inthe backbone of the variant BBPI generates a modified variant BBPI thathas a greater production yield that the unmodified variant BBPI. In someembodiments, BBPIs that comprise a combination of two, three, four,five, six, seven or eight amino acid substitutions have a greaterproduction yield than the unmodified precursor BBPI. Thus, the inventionprovides for modified variant BBPIs that comprise any one combination ofthe amino acid substitutions described above and that have greaterproduction yield (PY) than the unmodified precursor variant BBPIs. Inyet other embodiments, the invention provides for modified variant BBPIsthat comprise any one combination of the amino acid substitutionsdescribed above and that have greater trypsin inhibitory activity andgreater production yield than the TIA and PY of the unmodified precursorvariant BBPIs.

In some embodiments, the modified variant BBPIs that have greater TIAand PY than the corresponding precursor unmodified BBPIs each comprisefurther comprise a peptide insert that is positioned at the N-terminusof the modified variant BBPI. In some embodiments, the peptide insertcomprises a sequence of between 1 and 15 amino acids. In otherembodiments, the peptide insert comprises a sequence between 5 and 10amino acids. In some embodiments, the peptide insert comprises thepeptide of SEQ ID NO:389. Examples of modified variant BBPIs include themodified variant 4D13BBIt-AV of SEQ ID NO:390, and the modified variantBBIt-AV-4D13-13I-29P-40K-50T-52A of SEQ ID NO: 413.

One measure of enhancement in production yield can be determined as theincrease in the level of free BBPI following cleavage of the BBPI fromthe C-terminus of the BCE core. As described above, in some embodiments,the BBPI fusion proteins can be cleaved using proteases or by chemicalmeans. In other embodiments, the BBPI can be cleaved from the BBPIfusion protein by acid/heat treatment, which cleaves the A-P bondspresent in the CBD linker that joins the BCE to the BBPI. In yet otherembodiments, the BBPI fusion proteins can be cleaved using glutamylendopeptidase I from B. licheniformis. In some embodiments, theenhancement in production yield of a modified variant BBPI is measuredas the increase in the level of free modified variant BBPI followingactivation of the fusion BBPI and following acid heat treatment of theBBPI fusion protein, when compared to the level of free unmodifiedprecursor BBPI that was subjected to the same treatment. In otherembodiments, the enhancement in production yield of a modified variantBBPI is measured as the increase in the level of free modified variantBBPI without activation of the fusion BBPI and following acid heattreatment of the BBPI fusion protein, when compared to the level of thecorresponding free unmodified precursor BBPI that was subjected to thesame treatment. In some embodiments, the free modified variant BBPI hasa greater production yield and a greater TIA than the correspondingprecursor BBPI.

In some embodiments, the production yield of the modified variant BBPIis greater than that of the corresponding unmodified precursor BBPI byat least about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%,about 3.0%, about 4.0%, about 5.0%, about 8.0%, about 10%, about 15%,about 20%, about 25%, about 30%, about 40%, about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 100% or more. In other embodiments, the production yield ofthe modified variant BBPI is greater than that of the correspondingunmodified precursor BBPI by at least about 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, and up to at least about 200%.

6. PERSONAL CARE COMPOSITIONS

6.0.1 Personal Care Compositions Comprising Modified Variant BBPIs

The present invention provides personal care compositions comprising atleast one modified variant BBPI for medical and nonmedical conditions.In some embodiments, the present invention provides personal carecompositions and the methods of their use. In some embodiments, theinvention provides personal care compositions for use in the skin care.In other embodiments, the personal care compositions are for use in haircare. In some embodiments, the personal care compositions for use inskin care include cosmetic compositions. In other embodiments, thepersonal care compositions of the invention are for use in the treatmentof various disorders.

As described in greater detail herein, the present invention providescompositions for use in numerous aspects of personal care, including butnot limited to hair and skin care, as well as cosmetics (e.g., make-up).For example, the present invention provides compositions that find usein daily personal care, skin care, sun care (e.g., sunscreens, as wellas tanners), hair care (e.g., shampoos, leave-on and/or rinse offconditioners, hair tonics, hair sprays, gels, foams, mousses, settingproducts, hair colorants, permanent formulations, other styling andcleaning products, etc.), after-sun care for skin, hair and lips, oralcare (e.g., toothpastes and gels, mouthwashes, rinses, etc.), bathing(e.g., washes, shower soaps, bath soaps, salts, pearls, etc.), skinlighteners, cleansing treatments for skin conditions (e.g., pimples,acne, skin toners, etc.), depilatories, wet wipes, deodorants,antiperspirants, facial masks, shaving (e.g., shaving creams, gels,etc.), after-shave, skin peeling (e.g., exfoliants), intimate careproducts (e.g., feminine hygiene products), personal fresheners, andfoot care. The present invention also provides compositions that finduse in cosmetics (e.g., foundations, mascara, eye shadows, eye liners,lipsticks, lip glosses, blushers, etc.). In addition, the presentinvention provides personal care compositions for use in ameliorating ahair condition associated with a disorder. In other embodiments, thepersonal care composition are for use in ameliorating a skin conditionassociated with a disorder. It is contemplated that the compositions ofthe present invention will find use various forms, including but notlimited to solids, liquids, colloidal suspensions, emulsions, oils,gels, aerosols, foams, powders, pump sprays, etc., as well as being usedin conjunction with items such as wet wipes, etc. Indeed, it iscontemplated that the present invention will find use in any suitableform for the intended use(s).

In some embodiments, the personal care composition comprises a modifiedvariant BBPI comprising a variant peptide and at least one amino acidsubstitution, as described in section 5.4.1 above. In other embodiments,the personal care composition comprises a modified variant BBPIcomprising a variant peptide and a combination of amino acidsubstitutions, as described in section 5.4.2 above. In some embodiments,the personal care composition comprises a modified variant BBPI in whichthe equivalent chymotrypsin loop is a VEGF-binding peptide. In otherembodiments, the personal care composition comprises a modified variantBBPI in which the equivalent chymotrypsin loop is an FGF5 bindingpeptide. In other embodiments, the personal care composition comprises amodified variant BBPI in which the equivalent chymotrypsin loop is aTGFβ binding peptide. In yet other embodiments, the personal carecomposition comprises a modified variant BBPI in which the equivalentchymotrypsin loop is a TNFα binding peptide. In some embodiments, themodified variant BBPI comprises from about 0.0001 weight percent toabout 5 weight percent of the personal care composition, while inalternative embodiments, the modified variant BBPI comprises from about0.001 weight percent to about 0.5 weight percent of the personal carecomposition, and in yet additional embodiments, the modified variantBBPI comprises from about 0.01 weight percent to about 1 weight percentof the personal care composition.

6.0.2 Personal Care Compositions Comprising Modified Variant VEGF-BBPIs

VEGF plays a central role in promoting angiogenesis as well asinfluencing diverse cell functions including cell survival,proliferation and the generation of nitric oxide and prostacyclin(Zachary et al., Cardiovasc Res, 49: 568-81 [2001]). The recognition ofVEGF as a primary stimulus of angiogenesis in pathological conditionshas led to various attempts to block VEGF activity. Inhibitory anti-VEGFreceptor antibodies, soluble receptor constructs, antisense strategies,RNA aptamers against VEGF and low molecular weight VEGF receptortyrosine kinase (RTK) inhibitors have all been proposed for use ininterfering with VEGF signaling (See, Siemeister et al., [1998]). Infact, monoclonal antibodies against VEGF have been shown to inhibithuman tumor xenograft growth and ascites formation in mice (See, Kim etal., [1993]; Asano et al., [1998]; Mesiano et al., [1998]; Luo et al.,[1998a] and [1998b]; and Borgstrom et al., [1996] and [1998]).

Angiogenesis, involving VEGF and RTKs is not only involved in cancerdevelopment, as many other diseases or conditions affecting differentphysiological systems are angiogenesis-dependent, such as arthritis andatherosclerotic plaques (bone and ligaments), diabetic retinopathy,neovascular glaucoma, macular degeneration, ocular herpes, trachoma andcorneal graft neovascularization (eye) and angiofibroma. VEGF expressionis upregulated in the hyperplastic epidermis of psoriasis patients(Detmar and Yeo et al. [1995]), and in other skin diseases characterizedby enhanced angiogenesis including, scleroderma, rosacea, hemangioma,contact dermatitis, and hypertrophic scarring of the skin. Targetedoverexpression of VEGF in the epidermis of transgenic mice was reportedto result in enhanced skin vascularization with equal numbers oftortuous and leaky blood vessels (See e.g., Brown et al., [1998]). Also,chronic synthesis of VEGF in mouse skin leads to the firsthistologically equivalent murine model of human psoriasis (Xia et al.,[2003]) that is reversible by binding agents specific for VEGF. Inaddition, ultraviolet radiation induces VEGF production in keratinocytesand enhances cutaneous angiogenesis (Kim et al., Soc. InvestigativeDermatol. 126:2697 [2006]; Blaudshun et al., FEBS Let. 474:195-200;Kosmadaki et al., FASEB J. 17:446-8 [2003]).

In addition, the expression of VEGF in the outer root sheath of murinehair follicles was found to be temporally and spatially associated withcapillary proliferation during anagen. Transgenic overexpression of VEGFin the outer root sheath increased perifollicular vascularization andled to accelerated hair growth following depilation and the growth oflarger hairs (Yano et al. J Clin Invest 107: 409-17 [2001]).

Thus, VEGF is involved in the vascularization associated with pathologicand non-pathologic conditions.

The invention provides for personal skin care and/or hair carecompositions that comprise a modified variant VEGF-BBPI. The VEGF-BBPIscomprised in the personal care compositions of the invention aremodified variant BBPIs in which the equivalent chymotrypsin loop of theprecursor scaffold of the VEGF-BBPI has been replaced by a VEGF variantpeptide, and which further comprise at least one amino acid substitutionas described in sections 5.4.1 and 5.4.2. In some embodiments of thepresent invention, binding of the modified variant VEGF-BBPI to VEGFprevents VEGF from increasing perifollicular vascularization andinhibits the VEGF from promoting hair growth. In other embodiments,binding of the modified variant VEGF-BBPI to VEGF prevents VEGF fromincreasing vascularization of the skin in a subject suffering from anangiogenic skin disorder e.g. hemangioma and lichen planus. In yet otherembodiments, binding of the modified variant VEGF-BBPI to VEGF preventsVEGF from promoting disregulated angiogenesis associated withinflammatory skin disorders including psoriasis, scleroderma, venousulcers, acne, rosacea, warts, eczema, and lymphangiogenesis. However, itis not intended that the present invention be limited to any particularmechanism.

In some embodiments, the personal care composition comprises a VEGF-BBPIin which the chymotrypsin loop is a VEGF-binding peptide chosen fromU.S. application Ser. Nos. 09/832,723 and 10/984,270, including peptidesACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ IDNO:459), DLYWW (SEQ ID NO:460), SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ IDNO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ IDNO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS(SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498),STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ IDNO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET(SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509),TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ IDNO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW(SEQ ID NO:518), and HLAPSAV (SEQ ID NO:519). In some other embodiments,the VEGF variant sequences include, but are not limited to VEGF-bindingpeptides disclosed in U.S. application Ser. No. 11/919,717, includingpeptides KYYLSWW (SEQ ID NO:520), WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQID NO:522), RYSLYYW (SEQ ID NO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW(SEQ ID NO:525), TKWPSYW (SEQ ID NO:226), TLWKSYW (SEQ ID NO:527),PLWPSYW (SEQ ID NO:528), RLWPSYW (SEQ ID NO:529), TLWPKYW (SEQ IDNO:530), KYDLYWW (SEQ ID NO;531), RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQID NO:533), DYKLYWW (SEQ ID NO:534), EYKLYWW (SEQ ID NO:535), andRYPLYWW (SEQ ID NO:536). In other embodiments, the VEGF binding peptideis chosen from SEQ ID NOS:9, 458, 459, 460, 468, 469, 470, 471, 472 and473.

The scaffold in which the variant VEGF peptide is introduced to replacethe chymotrypsin loop is chosen from the scaffolds of the soybeaninhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature andtruncated form thereof (SEQ ID NO:185), the inhibitor from Dolichosbiflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycinemax (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana)cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186),the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ IDNO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffoldof (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), theBBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SEID NO:642). In addition, any wild-type BBPI precursor scaffolds, such asthose disclosed by Prakash et al. (supra), may be used to generatevariant BBPI scaffolds. In some embodiments, the scaffold of theVEGF-BBPI is that of SEQ ID NO:187.

In some embodiments, the backbone of the modified variant VEGF-BBPIcomprises at least one amino acid substitution at least at one aminoacid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18,25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ IDNO:187, as described in section 5.4.1.

In other embodiments, the backbone of the modified variant VEGF-BBPIcomprises a combination of amino acid substitutions chosen from acombination two, three, four, five, six, seven or eight amino acidsubstitutions as recited above in section 5.4.2.

In other embodiments, the backbone of the modified variant VEGF-BBPIcomprises a combination of amino acid substitutions chosen from acombination of amino acid substitutions chosen from 13I-29P-50T-52A,13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L.

In some embodiments, the personal care compositions of the inventioncomprise a modified variant BBPI in which the equivalent chymotrypsinloop of the precursor scaffold is replaced with a VEGF variant peptidechosen from SEQ ID NOS:9, 458, 459, 460, 468, 469, 470, 471, 472 and473, wherein the scaffold is that of SEQ ID NO:187, and which comprisesa combination of amino acid substitutions chosen from 13I-40K-50T-52A,13I-25K-29P-52K, 13I-29P-40K-50T-52A, 13I-25R-27A-29P-31A-50K-52T,13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q,13I-25K-27A-29R-31A-40H-50R-52L. In some embodiments, the personal carecompositions comprise a VEGF-BBPI chosen from the VEGF-BBPIs of SEQ IDNOS:601, 602, 627, 628, 629, 630, 631, 643, 491, 632, 633, 634, 635 and636.

In some embodiments, the invention provides a personal care compositioncomprising a VEGF-BBPI that binds to VEGF. In alternative embodiments,the binding of the VEGF-BBPI to VEGF blocks the downstream activity ofVEGF. In some embodiments, the composition is capable of modulatingangiogenesis.

In some embodiments, the personal care composition comprises a modifiedvariant BBPI-VEGF for use in skin care. In some embodiments, the skincare compositions are for cosmetic use in improving the appearance ofskin. In other embodiments, the skin care compositions are fortherapeutic use in improving the appearance of skin in a subjectsuffering from a skin disorder. In some embodiments, the skin disorderis an angiogenic skin disorder. In additional embodiments, the skindisorder is at least one chosen from psoriasis, scleroderma, venousulcers, acne, rosacea, warts, eczema, hemangiomas and lymphangiogenesis.In some embodiments, the skin disorder is rosacea. In other embodiments,the skin disorder is psoriasis.

In one embodiment, the personal skin care composition comprising aVEGF-BBPI is chosen from skin creams, lotions, sprays, emulsions,colloidal suspensions, foams, aerosols, liquids, gels, sera, and solids.In another embodiment, the personal care composition is a skin carecomposition selected from moisturizing body washes, body washes,antimicrobial cleansers, skin protective creams, body lotions, facialcreams, moisturizing creams, facial cleansing emulsions, facial gels,facial sera, surfactant-based facial cleansers, facial exfoliating gels,anti-acne treatments, facial toners, exfoliating creams, facial masks,after shave balms, pre-shave balms, tanning compositions, skinlightening compositions, skin redness reduction compositions,sunscreens, depilatories, hair growth inhibitors, and radioprotectives.Radioprotectives are chosen from non-water-resistant sunscreens, verywater-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal care skin care composition comprising aVEGF-BBPI is comprises topically applied over-the-counter compositions,anti-fungal treatments, anti-acne treatments, skin protectants,sunscreens, deodorants, and antiperspirants. In other embodiments, theskin care composition is capable of lightening skin tone, reducingredness, preventing skin tone darkening or preventing color development.

The present invention also provides personal care compositions that arecosmetic compositions. In some preferred embodiments, the cosmeticcompositions comprising a VEGF-BBPI are chosen from pressed powderformulations and foundations. In some preferred embodiments, thecosmetic compositions comprise at least one pigment.

In yet additional embodiments, the makeup compositions are pressedpowder formulations selected from loose powders, blushes, and bronzingpowders. In still further embodiments, the makeup compositions arefoundations selected from water-in-oil foundations, water-in-siliconefoundations, oil-in-water foundations, anhydrous makeup sticks, andcream-to-powder foundations.

In some embodiments, the personal skin care compositions comprise amodified variant VEGF-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the VEGF-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the VEGF-BBPI is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In some other embodiments, the invention provides a personal carecomposition that comprises a modified variant VEGF-BBPI for use in haircare.

In some embodiments, the hair care compositions find use in inhibitinghair growth. In other embodiments, inhibition of hair growth compriseshair removal for treatment of at least one disease or condition forwhich decreased hair growth is desirable. In some embodiments,inhibition and/or removal comprises depilation. In some embodiments, thehair is selected from the group consisting of facial hair, leg hair, armhair, and torso hair.

In one embodiment, the hair care composition is selected from the groupconsisting of shampoos, conditioners, hair styling compositions, haircolorants, permanent wave formulations, creams, gels, mousses, sprays,emulsions, colloidal suspensions, liquids, foams, and solids. In someembodiments, the hair care composition further comprises aradioprotective. As described for the personal skin care compositionsthe radioprotective is a sunscreen chosen from non-water-resistantsunscreens, very water-resistant sunscreens, and water-in-siliconesunscreens. In other embodiments, the radioprotective is a sunscreenchosen from non-water-resistant sunscreens, very water-resistantsunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise amodified variant VEGF-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the VEGF-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the VEGF-BBPI is present inan amount of about 0.001% to about 1% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the personal care compositions of the inventionare for use in the treatment of various diseases associated withelevated levels of VEGF.

The present invention also provides methods for inhibiting hair growthof a subject, comprising the steps of providing the personal care VEGFhair care composition of the present invention; providing a subject tobe treated; and applying the composition to the subject in an area inwhich inhibition of hair growth is desired. In some embodiments, theinhibition of hair growth comprises inhibiting the growth of thesubject's hair, wherein the hair to be inhibited is selected from thegroup consisting of facial air, underarm hair, leg hair, torso hair, andarm hair, and head hair. In additional embodiments, the method forinhibiting hair growth using a VEGF hair care composition of theinvention comprises a personal care composition comprising a VEGF-BBPIhaving an amino acid sequence chosen from SEQ ID NOS SEQ ID NOS:601,602, 627, 628, 629, 630, 631, 643, 491, 632, 633, 634, 635 and 636.

In another embodiment, the invention provides a method for improving theappearance and/or condition of skin in a subject suffering from a skindisorder, comprising providing the personal care composition of thepresent invention; providing a subject to be treated; and applying thecomposition to the affected skin of the subject. In some embodiments,the skin disorder is chosen from psoriasis, venous ulcers, acne,rosacea, warts, eczema, hemangiomas, cutaneous lichen planus, andlymphangiogenesis, etc. In some particularly preferred embodiments, theskin disorder is rosacea. In other embodiments, the skin disorder ispsoriasis. In additional embodiments, the method for improving theappearance and/or condition of skin in a subject suffering from a skindisorder using a VEGF skin care composition of the invention comprises apersonal care composition comprising a VEGF-BBPI having an amino acidsequence chosen from SEQ ID NOS:601, 602, 627, 628, 629, 630, 631, 643,491, 632, 633, 634, 635 and 636.

6.0.3 Personal Care Compositions Comprising Modified Variant FGF-5-BBPIs

The Fibroblast Growth Factor (FGF) family is a superfamily of growthfactors containing at least 23 members, many of which are potentregulators of cell proliferation, differentiation and cell function. Allof the FGFs have a conserved 120 amino acid core. Members of the familyshare conserved cysteine residues and 30-50% sequence homology at theamino acid level. The molecular weight of the FGFs ranges from 7 kDa forFGF-1 to 38 kDa for FGF-5. The length of the proteins is from 60 aminoacids in the case of an FGF-1 splice variant to 288 amino acids forFGF-2. Binding to heparin is an essential step required for an FGFfactor to interact with cell surface receptors. FGF5 is a secretedsignaling protein consisting of 268 amino acids with a 17 amino acidsignal sequence and a 251 amino acid mature peptide. The human gene alsogives rise to a glycosylated alternate splice form that is 18 kDa insize and 123 amino acid in length. The murine homologue of FGF-5 wascloned and found to be 84% homologous to the human protein at the aminoacid sequence level. Human FGF-5 consists of three exons and maps tochromosome 4q21 and cross-reacts with murine FGF-5.

Formation of hair follicles involves a complex series of steps: growth(anagen), regression (catagen), rest (telogen) and shedding (exogen).FGF-5 has been implicated as one of the major drivers of the transitionfrom anagen to catagen in the hair cycle. Expression of FGF-5 isdetected in hair follicles from wild-type mice and is localized to theouter root sheath during the anagen phase. Mice homozygous for apredicted null allele of FGF-5, fgf5neo, have abnormally long hair (See,Hebert et al., Cell 78: 1017-25 [1994]). The phenotype appears identicalto that of mice homozygous for the spontaneous mutation angora (go).Recently, partial FGF-5 sequences, FGF5S, thought to compete with FGF-5in binding to the receptor have been identified, (See, Ito et al., J.Cell Physiol., 197:272-83 [2003]).

The invention provides personal hair care compositions that comprises atleast one modified variant FGF-BBPI for use in promoting hair growthand/or preventing hair loss. In one embodiment, the invention providesFGF-BBPI compositions for personal skin care. In other embodiments, theinvention provides FGF-BBPI compositions for hair care. The FGF-BBPIscomprised in the personal care compositions of the invention aremodified variant BBPIs in which the equivalent chymotrypsin loop of theprecursor scaffold of the FGF-5-BBPI has been replaced by an FGF-5variant peptide, and which further comprise at least one amino acidsubstitution as described in sections 5.4.1 and 5.4.2. In the presentinvention, binding of the modified variant FGF-BBPI to FGF-5 preventsFGF-5 from interacting with its cognate receptor and inhibits transitionfrom the anagen to the catagen promoting hair growth and preventing hairloss. However, it is not intended that the present invention be limitedto any particular mechanism.

In some embodiments, personal care composition comprises at least oneFGF-BBPI in which the chymotrypsin loop is an FGF-5 variant peptidechosen from CACRTQPYPLCF (MM007; SEQ ID NO:430), CICTWIDSTPC (PS2; SEQID NO:431), CYGLPFTRC (SEQ ID NO:537), CEEIWTMLC (SEQ ID NO:538),CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ ID NO:540), CTLWNRSPC (SEQ IDNO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC (SEQ ID NO:543), TNIDSTP(SEQ ID NO:544), HLQTTET (SEQ ID NO:545), SLNNLTV (SEQ ID NO:546),TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ ID NO:548), LRILANK (SEQ IDNO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQ ID NO:551), TNIDSTP (SEQID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP (SEQ ID NO:554), TNIDSTP(SEQ ID NO:555), HLQTTET (SEQ ID NO:556), PLGLCPP (SEQ ID NO:557),GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ ID NO:559), HLQTTET (SEQ IDNO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQ ID NO:562), RTQPYPL (SEQID NO:670) and TWIDSTP (SEQ ID NO:671). In other embodiments, the FGFvariant peptide is chosen from SEQ ID NOS: SEQ ID NOS:430, 431, 670 and671.

The scaffold in which the variant FGF peptide is introduced to replacethe chymotrypsin loop is chosen from the scaffolds of the soybeaninhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature andtruncated form thereof (SEQ ID NO:185), the inhibitor from Dolichosbiflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycinemax (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana)cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186),the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ IDNO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffoldof (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), theBBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SEID NO:642). In addition, any wild-type BBPI precursor scaffolds, such asthose disclosed by Prakash et al. (supra), may be used to generatevariant BBPI scaffolds. In some embodiments, the scaffold of theVEGF-BBPI is that of SEQ ID NO:187.

In some embodiments, the backbone of the modified variant FGF-BBPIcomprises at least one amino acid substitution at least at one aminoacid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18,25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ IDNO:187, as recited in section 5.4.1. In other embodiments, the backboneof the modified variant FGF-BBPI comprises a combination of amino acidsubstitutions chosen from a combination two, three, four, five, six,seven or eight amino acid substitutions as recited above in section5.4.2. In other embodiments, the backbone of the modified variantFGF-BBPI comprises a combination of amino acid substitutions chosen froma combination of amino acid substitutions chosen from 13I-29P-50T-52A,13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L. Insome embodiments, the combination of substitutions is 13I-29P-50T-52A.

In some embodiments, the personal care compositions of the inventioncomprise an FGF-BBPI in which the equivalent chymotrypsin loop of theprecursor scaffold is replaced with an FGF variant peptide chosen fromSEQ ID NOS:433 and 434, wherein the scaffold is that of SEQ ID NO:187,and which comprises the combination of amino acid substitutions13I-40K-50T-52A. Thus, in some embodiments, the personal carecompositions comprise a FGF-BBPI chosen from the FGF-BBPIs of SEQ IDNOS:439 and 441.

In some embodiments, the invention provides a personal care compositioncomprising a FGF-BBPI that binds to FGF. In alternative embodiments, thebinding of the FGF-BBPI to FGF blocks the downstream activity of FGF. Insome embodiments, the composition is capable of promoting hair growth.

In some embodiments, the personal care composition comprising a modifiedvariant BBPI-FGF is for use in skin care. In some embodiments, the skincare compositions are cosmetic compositions for use in promoting hairgrowth. In some embodiments, the skin care compositions are for use inpromoting hair growth in a subject suffering from a disease or conditionthat involves hair loss. In some of these embodiments, the disease orcondition is at least one selected from the group consisting ofinflammatory alopecias, pseudopelade, scleroderma, tick bites, lichenplanus, psoriasis, lupus, seborrheic dermatitis, loose hair syndrome,hemochromatosis, androgenic alopecia, alopecia areata, cancer,conditions that affect defective hair fiber production, andenvironmental factors that affect hair production. In a preferredembodiment, the disease is androgenic alopecia or alopecia areata.

In one embodiment, the personal care composition comprising an FGF-BBPIis a skin care composition is chosen from skin creams, lotions, sprays,emulsions, colloidal suspensions, foams, aerosols, liquids, gels, sera,and solids. In another embodiment, the personal care composition is askin care composition selected from moisturizing body washes, bodywashes, antimicrobial cleansers, skin protective creams, body lotions,facial creams, moisturizing creams, facial cleansing emulsions, facialgels, facial sera, surfactant-based facial cleansers, facial exfoliatinggels, anti-acne treatments, facial toners, exfoliating creams, facialmasks, after shave balms, pre-shave balms, tanning compositions, skinlightening compositions, skin redness reduction compositions,sunscreens, depilatories, hair growth inhibitors, and radioprotectives.Radioprotectives are chosen from non-water-resistant sunscreens, verywater-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal skin care composition comprising anFGF-BBPI is a skin care composition comprising topically appliedover-the-counter compositions, anti-fungal treatments, anti-acnetreatments, skin protectants, sunscreens, deodorants, andantiperspirants.

In some embodiments, the personal skin care compositions comprise amodified variant VEGF-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the VEGF-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the VEGF-BBPI is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the invention provides a personal care compositioncomprising a modified variant FGF-BBPI for use in hair care.

In some embodiments, the hair care compositions find use in promotinghair growth. In other embodiments, modulation comprises promoting hairgrowth in a subject suffering from a disease or condition that involveshair loss. In some of these embodiments, the disease or condition is atleast one selected from the group consisting of inflammatory alopecias,pseudopelade, scleroderma, tick bites, lichen planus, psoriasis, lupus,seborrheic dermatitis, loose hair syndrome, hemochromatosis, androgenicalopecia, alopecia areata, cancer, conditions that affect defective hairfiber production, and environmental factors that affect hair production.In a preferred embodiment, the disease is androgenic alopecia oralopecia areata.

In one embodiment, the hair care composition is selected from the groupconsisting of shampoos, conditioners, hair styling compositions, haircolorants, permanent wave formulations, creams, gels, mousses, sprays,emulsions, colloidal suspensions, liquids, foams, and solids. In someembodiments, the hair care composition further comprises aradioprotective. As described for the personal skin care compositionsthe radioprotective is a sunscreen chosen from non-water-resistantsunscreens, very water-resistant sunscreens, and water-in-siliconesunscreens. In other embodiments, the radioprotective is a sunscreenchosen from non-water-resistant sunscreens, very water-resistantsunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise amodified variant FGF-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the FGF-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the FGF-BBPI is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the personal care FGF-BBPI compositions of theinvention are for use in the treatment of various diseases associatedwith elevated levels of FGF.

The present invention also provides methods for promoting hair growth ofa subject, comprising the steps of providing the personal care FGFcomposition of the present invention; providing a subject to be treated;and applying the composition to the subject in an area in whichpromotion of hair growth is desired. In some embodiments, the FGFcomposition is a skin care composition. In other embodiments, the FGFcomposition is a hair composition. In some embodiments, promotion ofhair growth comprises promoting the growth of the subject's hair,wherein the hair growth to be promoted is chosen from facial air,underarm hair, leg hair, torso hair, and arm hair, and head hair. Inadditional embodiments, the method for promoting hair growth using anFGF hair care composition of the invention comprises applying a personalcare composition comprising an FGF-BBPI having an amino acid sequencechosen from SEQ ID NOS:439 and 441.

6.0.4 Personal Care Compositions Comprising Modified Variant TGFβ-BBPIs

Proteins of the Transforming Growth Factor-β (TGFβ) family aresynthesized by almost all cells. The TGFβs are a group of stable,multifunctional polypeptide growth factors whose activities include,among other things, context-specific inhibition and stimulation of cellproliferation, control of the extracellular matrix, degradation andcontrol of mesenchymal-epithelial interactions during embryogenesis,mediation of cell and tissue responses to injury, control ofcarcinogenesis and modulation of immune responses. TGFβ-1 is synthesizedby virtually all cells (with only a few exceptions). TGFβ-1 has beenfound in the highest concentration in human platelets and mammalianbone. TGFβ-1 has many functions including suppression of cellproliferation, enhancement of extracellular matrix deposition andphysiological immunosuppression. TGFβ-1 has also been determined to bebiologically active in hair follicle development. Human TGFβ-1 is a 25.0kDa protein with subunits that contain approximately 112 amino acids persubunit. Two different receptor proteins are involved in TGFβ-1 bindingand signaling, namely TGF-RβII and TGF-RβI. TGFβ-2 is expressed in avariety of cells, including keratinocytes, fibroblasts, osteoclasts,thymic epithelium, skeletal muscle cells, prostatic epithelium,bronchial epithelium, neurons and astrocytes, visceral smooth muscle,macrophages and various other cells. TGFβ-2 has many fundamentalactivities, including function as a growth inhibitor for most cells, anenhancer for deposition of the extracellular matrix, andimmunosuppression. The mature region is 71% identical to TGFβ-1, 80%identical to TGFβ-3, and 97% identical to the mouse homologue of thesame protein at the amino acid level. TGFβ-2 dimerizes with formation ofdisulfide bonds between the ‘pro’ regions and disulfide bonds betweenthe mature regions. TGFβ-2 is synthesized as a pre-procytokine with a 19amino acid signal sequence, a 283 pro-region and a 112 mature amino acidsegment. The receptor for TGFβ-2 forms a heterotetrameric complex of twotype I signal-transduction receptors and two type II ligand-bindingreceptors.

Formation of hair follicles involves a complex series of steps: growth(anagen), regression (catagen), rest (telogen) and shedding (exogen)(See, Sten and Paus, Physiol. Rev, Exp. Dermatol., 8:229-233 [1999]).TGFβs have been implicated as one of the major drivers of the transitionfrom anagen to catagen in the hair cycle (See e.g., Foitzik et al, FASEBJ., 5:752-760 [2000]; and Soma et al. J. Infect. Dis., J. Invest.Dermatol., 118:993-9997 [2002]), and TGFβ2 is both a required andsufficient inducer of murine hair follicle morphogenesis (See, Foitziket al., Develop. Biol., 212:278-289 [1999]). Conditional TGFβ-1expression in transgenic mice demonstrates that one can induce alopeciareversibly (See, Liu et al., Proc. Natl. Acad. Sci. USA 98:9139-9144[2001]). In addition, TGFβ-1 mutants have been associated with the delayof catagen onset in mice (See, Foitzik et al, [2000], supra). Recently,it has been shown that catagen can be delayed through the use of TGFβ-2antibodies (See, Soma et al., [2002], supra). Finally, androgens thatinduce TGFβ-1 production in balding dermal papilla cells can inhibitepithelial cell growth (Inui et al., FASEB J., 14:1967-1969 [2002]).

TGFβ is also a potent stimulus of connective tissue accumulation, and isimplicated in the pathogenesis of scleroderma and other fibroticdisorders (Blobe et al., N Engl J Med 342:1350-1358 [2000]). Sclerodermais a chronic autoimmune disease characterized by early inflammation andvascular injury, followed by progressive fibrosis of the skin and otherorgans (Kissin and Korn, Rheum Dis Clin North Am 29:351-369 [2003]). Themost evident symptom is usually the hardening of the skin and associatedscarring.

TGFβ has also been implicated in the formation of skin tumors (Li etal., Molecular Carcinogenesis 45:389-396 [2006]). The TGFβ signalingpathway is one of the most important mechanisms in the maintenance ofepithelial homeostasis. Alterations leading to either the repression orenhancement of this pathway have been shown to affect cancerdevelopment. Although TGFβ inhibits growth of normal epithelial cells,it is paradoxically overexpressed in many epithelial cancers. It hasbeen postulated that TGFβ acts as a tumor suppressor at the early stagesof carcinogenesis, but overexpression of TGFβ at late stages ofcarcinogenesis may be a critical factor for tumor invasion andmetastasis.

The invention provides personal care compositions that comprise amodified variant TGF-BBPI. The TGFβ-BBPI comprised in the personal carecompositions of the invention is a modified variant TGFβ-BBPI in whichthe chymotrypsin loop of the precursor scaffold of the TGF-BBPI has beenreplaced by a TGFβ variant peptide, and which further comprise at leastone amino acid substitution as described in sections 5.4.1 and 5.4.2. Insome embodiments, binding of the modified variant TGF-BBPI to TGFβ1and/or TGFβ2 prevents TGFβ1 and/or TGFβ2 from interacting with itscognate receptor and inhibits transition from the anagen to the catagento promote hair growth and preventing hair loss. In other embodiments,binding of the modified variant TGF-BBPI to TGFβ1 and/or TGFβ2 preventsTGFβ1 and/or TGFβ2 from interacting with its cognate receptor to preventfibrosis of the skin that is characteristic of scleroderma. In otherembodiments, binding of the modified variant TGF-BBPI to TGFβ1 and/orTGFβ2 prevents TGFβ1 and/or TGFβ2 from interacting with its cognatereceptor to prevent progression of benign to malignant skin lesions.However, it is not intended that the present invention be limited to anyparticular mechanism.

In some embodiments, the TGFβ variant peptide comprised in the TGFβ-BBPIis chosen from CLCPENINVLPCN (PEN3; SEQ ID NO:436), CICKHNVDWLCF(MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ ID NO:438), CVTTDWIEC(SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC (SEQ ID NO:565),QSACIVYYVGRKPKVECASSD (SEQ ID NO:566), QSACILYYIGKTPKIECASSD (SEQ IDNO:567), QSACILYYVGRTPKVECASSD (SEQ ID NO:568),acetyl-LCPENDNVSPCY-cohn2 (SEQ ID NO:569), KHNVRLL (SEQ ID NO:570),NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQ ID NO:572), RGPAHSL (SEQ IDNO:573), NSLAERR (SEQ ID NO:574), HPLASPH (SEQ ID NO:575), QPWNKLK (SEQID NO:576), AWLr/Mipy (SEQ ID NO:577), PTKPAQQ (SEQ ID NO:578), PSLNRPQ(SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ ID NO:581),ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC (SEQ IDNO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647), CNGKSKLAC(SEQ ID NO:648), PENINVLP (SEQ ID NO;672), KHNVDWL (SEQ ID NO:673), andWTQHIHNC (SEQ ID NO:674). SEQ ID NOS:644-648 are TGFβ1-binding peptides,while SEQ ID NOS:463, 437, 438, 563-582 are TGFβ2-binding peptides.

In other embodiments, the TGFβ variant peptide is chosen from SEQ IDNOS: SEQ ID NOS:436, 437 and 438.

The scaffold in which the variant TGF peptide is introduced to replacethe equivalent chymotrypsin loop is chosen from the scaffolds of thesoybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature andtruncated form thereof (SEQ ID NO:185), the inhibitor from Dolichosbiflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycinemax (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana)cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186),the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ IDNO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffoldof (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), theBBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SEID NO:642). In addition, any wild-type BBPI precursor scaffolds, such asthose disclosed by Prakash et al. (supra), may be used to generatevariant BBPI scaffolds. In some embodiments, the scaffold of theVEGF-BBPI is that of SEQ ID NO:187.

In some embodiments, the backbone of the modified variant TGFβ-BBPIcomprises at least one amino acid substitution at least at one aminoacid position chosen from positions equivalent to 1, 4, 5, 11, 13, 18,25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of the variant BBI of SEQ IDNO:187, as recited in section 5.4.1.

In other embodiments, the backbone of the modified variant TGFβ-BBPIcomprises a combination of amino acid substitutions chosen from acombination two, three, four, five, six, seven or eight amino acidsubstitutions as recited above in section 5.4.2.

In other embodiments, the backbone of the modified variant TGFβ-BBPIcomprises a combination of amino acid substitutions chosen from acombination of amino acid substitutions chosen from 13I-29P-50T-52A,13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L. Insome embodiments, the combination of substitutions is 13I-29P-50T-52A.In some embodiments, the personal care compositions of the inventioncomprise a BBPI in which the equivalent chymotrypsin loop of theprecursor scaffold is replaced with a TGFβ variant peptide chosen fromSEQ ID NOS: 436, 437, 438, 672, 673, and 674, wherein the scaffold isthat of SEQ ID NO:187, and which comprises the combination of amino acidsubstitutions 13I-29P-50T-52A. In some embodiments, the personal carecompositions comprise a FGF-BBPI chosen from the TGFβ-BBPIs of SEQ IDNOS:443, 445 and 447.

In some embodiments, the invention provides a personal care compositioncomprising a TGFβ-BBPI that binds to TGFβ1 and/or TGFβ2. In alternativeembodiments, the binding of the TGFβ-BBPI to TGFβ blocks the downstreamactivity of TGFβ. In some embodiments, the composition is capable ofpromoting hair growth.

In some embodiments, the personal care composition comprising a modifiedvariant TGF-BBPI is for use in skin care. In some embodiments, the skincare compositions are cosmetic compositions for use in promoting hairgrowth.

In some embodiments, the skin care compositions are for use in promotinghair growth in a subject suffering from a disease or condition thatinvolves hair loss. In some of these embodiments, the disease orcondition is at least one selected from the group consisting ofinflammatory alopecias, pseudopelade, scleroderma, tick bites, lichenplanus, psoriasis, lupus, seborrheic dermatitis, loose hair syndrome,hemochromatosis, androgenic alopecia, alopecia areata, cancer,conditions that affect defective hair fiber production, andenvironmental factors that affect hair production. In a preferredembodiment, the disease is androgenic alopecia or alopecia areata.

In other embodiments, the personal care composition comprising amodified variant TGF-BBPI is for use in improving the appearance and/orcondition of the skin of a subject suffering from scleroderma. In yetother embodiments, the personal care composition comprising a modifiedvariant TGF-BBPI is for use in improving the appearance and/or conditionof the skin of a subject suffering from skin cancer.

In one embodiment, the personal care composition comprising a TGFβ-BBPIis a skin care composition is chosen from skin creams, lotions, sprays,emulsions, colloidal suspensions, foams, aerosols, liquids, gels, sera,and solids. In another embodiment, the personal care composition is askin care composition selected from moisturizing body washes, bodywashes, antimicrobial cleansers, skin protective creams, body lotions,facial creams, moisturizing creams, facial cleansing emulsions, facialgels, facial sera, surfactant-based facial cleansers, facial exfoliatinggels, anti-acne treatments, facial toners, exfoliating creams, facialmasks, after shave balms, pre-shave balms, tanning compositions, skinlightening compositions, skin redness reduction compositions,sunscreens, depilatories, hair growth inhibitors, and radioprotectives.Radioprotectives are chosen from non-water-resistant sunscreens, verywater-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal care composition comprising a TGFβ-BBPIis a skin care composition comprising topically applied over-the-countercompositions, anti-fungal treatments, anti-acne treatments, skinprotectants, sunscreens, deodorants, and antiperspirants.

The present invention also provides personal care compositions that arecosmetic compositions. In some preferred embodiments, the cosmeticcompositions are selected from mascaras, pressed powder formulations,and foundations. In some preferred embodiments, the makeup compositionscomprise at least one pigment.

In some preferred embodiments, the makeup composition comprising atleast one pigment is a mascara selected from non-waterproof mascaras,waterproof mascaras, volumizing mascaras, lengthening mascaras, curlingmascaras, anhydrous waterproof mascaras, water-based mascaras, andeyelash or eyebrow treatments.

In yet additional embodiments, the makeup compositions are pressedpowder formulations selected from loose powders, blushes, eye shadows,and bronzing powders. In still further embodiments, the makeupcompositions are foundations selected from water-in-oil foundations,water-in-silicone foundations, oil-in-water foundations, anhydrousmakeup sticks, and cream-to-powder foundations.

In some embodiments, the personal skin care compositions comprise amodified variant TGF-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the TGF-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the TGF-BBPI is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the invention provides a personal care compositionthat comprises a modified variant TGF-BBPI for use in hair care. In someembodiments, the hair care composition is for use in promoting hairgrowth.

In some embodiments, the hair care compositions find use in promotinghair growth. In other embodiments, modulation comprises promoting hairgrowth in a subject suffering from a disease or condition that involveshair loss. In some of these embodiments, the disease or condition is atleast one selected from the group consisting of inflammatory alopecias,pseudopelade, scleroderma, tick bites, lichen planus, psoriasis, lupus,seborrheic dermatitis, loose hair syndrome, hemochromatosis, androgenicalopecia, alopecia areata, cancer, conditions that affect defective hairfiber production, and environmental factors that affect hair production.In a preferred embodiment, the disease is androgenic alopecia oralopecia areata.

In one embodiment, the hair care composition is selected from the groupconsisting of shampoos, conditioners, hair styling compositions, haircolorants, permanent wave formulations, creams, gels, mousses, sprays,emulsions, colloidal suspensions, liquids, foams, and solids. In someembodiments, the hair care composition further comprises aradioprotective. As described for the personal skin care compositionsthe radioprotective is a sunscreen chosen from non-water-resistantsunscreens, very water-resistant sunscreens, and water-in-siliconesunscreens. In other embodiments, the radioprotective is a sunscreenchosen from non-water-resistant sunscreens, very water-resistantsunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise amodified variant TGFβ-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the TGFβ-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the TGFβ-BBPI is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the personal care TGF-BBPI compositions of theinvention are for use in the treatment of various diseases associatedwith elevated levels of TGFβ1 and/or TGFβ2.

The present invention also provides methods for promoting hair growth ofa subject, comprising the steps of providing the personal care TGFcomposition of the present invention; providing a subject to be treated;and applying the composition to the subject in an area in whichpromotion of hair growth is desired. In some embodiments, the TGFcomposition is a skin care composition. In other embodiments, the FGFcomposition is a hair composition. In some embodiments, promotion ofhair growth comprises promoting the growth of the subject's hair,wherein the hair growth to be promoted is chosen from facial air,underarm hair, leg hair, torso hair, and arm hair, and head hair. Inadditional embodiments, the method for promoting hair growth using anTGF hair care composition of the invention comprises applying a personalcare composition comprising a TGF-BBPI having an amino acid sequencechosen from SEQ ID NOS:443, 445 and 447.

In another embodiment, the invention provides a method for improving theappearance and/or condition of skin in a subject suffering from a skindisorder, comprising providing the personal skin care composition of thepresent invention; providing a subject to be treated; and applying thecomposition to the affected skin of the subject. In some embodiments,the skin disorder is chosen from psoriasis, scleroderma and skin cancer.In additional embodiments, the method for improving the appearanceand/or condition of skin in a subject suffering from a skin disorderusing a TGF skin care composition of the invention comprises a personalcare composition comprising a TGF-BBPI having an amino acid sequencechosen from SEQ ID NOS: 443, 445 and 447.

6.0.5 Personal Care Compositions Comprising Modified Variant TNFα-BBPIs

Proteins of the Tissue Necrosis Factor (TNF) family are members of alarge cytokine family. Tissue Necrosis Factor alpha (TNFα) is aprototypic member of the TNF family of ligands. It is produced byvarious immune cells, and plays a key role in immune-mediatedinflammatory diseases.

TNFα is synthesized by T-lymphocytes, B cells, synoviocytes,fibroblasts, and macrophages initially as a 26 kD protein. Later it iscleaved by TNFα converting enzyme (the metalloproteinase, ADAMS 17) intoa monomeric 17-kD molecule. Three of these molecules form, underphysiologic conditions, a non-covalently bound, cone-shaped homotrimerthat cross-links membrane-bound receptors that exist in 2 isoforms: TNFreceptor I (TNF-RI) and TNF receptor II (TNF-RII). Its natural functionis to stimulate the recruitment of neutrophils and monocytes to sites ofinfection and to activate these cells to eradicate microbes, exertingits function by binding to its corresponding TNF receptor on the surfaceof numerous cell types. It activates the vascular endothelium locally,causing vasodilation and increased permeability. Vascular endotheliumadhesion molecules (ICAM-1, VCAM-1, E-selectin) and MHC class II areupregulated leading to recruitment of proinflammatory cells, andinduction of immunoglobulins, and complement. Platelets become activatedand “stickier,” causing small vessel occlusion, and containment ofinfections. In addition, it induces macrophages and endothelial cells tosecrete chemokines and promotes apoptosis of target cells. TNFα has apotent paracrine function, inducing (via a NFκB-mediated mechanism) thesecretion of pro-inflammatory cytokines such as IL-1, IL-6, and GM-CSF,and also stimulates the production of various chemokines, includingRANTES, IL-8, MCP-1, and MIP-1α. Finally, TNFα plays a role inangiogenesis, which is critical to the growth and propagation of therheumatoid synovium.

Immune-mediated inflammatory diseases (IMIDs) are triggered by anabnormal production of pro-inflammatory cytokines, including TNFα. Thesediseases include: rheumatoid arthritis (RA), inflammatory bowel disease(IBD) such as Crohn's disease, chronic plaque psoriasis, psoriaticarthritis, vasculitis, and ankylosing spondylitis.

New developments in the treatment of immune-mediated inflammatorydiseases have arisen from basic research in cytokine expression andsignaling that identified two key players in the pathophysiology ofseveral diseases in this category: TNFα and interleukin interleukin-1(IL-1). For example, both cytokines have been found to be elevated inthe serum, synovium, and synovial fluid of patients with RA. Moreover,TNFα and IL-1 are capable of inducing and augmenting joint damage inexperimental models of arthritis. Such findings led to the developmentof strategies to block/antagonize their effects, and specific targetingby biological agents has become possible in the recent years. This firstgeneration of products includes monoclonal antibodies (mAb) and solublereceptor fusion proteins, all acting to compete with receptor forbinding of the cytokine.

To date, the US FDA has approved three TNFα inhibitors/blocking agentsfor clinical use in the treatment of several IMIDs, including psoriasisand psoriatic arthritis. They are: Etanercept (Enbrel®, Amgen-Wyeth), afully human chimeric protein of TNF receptor II fused to the Fccomponent of human IgG1, Infliximab (Remicade®, Centocor), a chimericmonoclonal antibody, and Adalimumab (Humira®, Abbott) a fully human IgG1anti-TNFα monoclonal antibody. The proven initial efficacy and safetyprofile of these agents has to be weighed against the concerns fordeclining efficacy over time, the high cost of these biopharmaceuticalagents and the limitations imposed by the current needle delivery forthese agents.

An animal model developed by Boyman and colleagues (Boyman et al., J.Exp. Med. 199:731-736 [2004]) demonstrated the essential role forresident T-cells and TNFα in the spontaneous development of psoriasis.In this model, symptomless pre-psoriatic human skin lesions are graftedonto transgenic AGR129 mice. The group showed that blocking of T cellslead to inhibition of psoriasis development. It also showed thatapplication of neutralizing anti-human TNFα monoclonal antibody(Infliximab, i.v.) or TNF receptor fusion protein (Etanercept, s.c.) ledto dramatic inhibition of psoriatic phenotype development. These resultshave served to support the notion that TNFα antagonists have a directeffect in IMIDs disease development.

Unfortunately, more than one third of patients suffering from any of theapproved IMIDs indications do not benefit clinically from anti-TNFtreatment (Wong et al., Clin. Immunol. 126:121-136 [2008]). Effortscontinue in the development of novel therapeutics to address thismedical area. There are a number of “smart” TNFα antagonists currentlyin clinical trials, including: various anti-TNF monoclonal antibodies,pegylated antibody fragments or truncated TNF receptor molecules. Inaddition, a small-molecule inhibitor that promotes TNFα subunitdisassembly of the trimeric cytokine has shown promising in vitroresults (He et al., Science 310:1022-1025 [2005]) but no furtherclinical development has been reported.

Psoriasis is a common skin disorder that affects approximately 2.8percent of the population (Linden and Weinstein Am. J. Med. 107:595-605[1999]). An estimated 4.5 million people in the US suffer from psoriasisand 1.5 million have moderate to severe plaque psoriasis. The disease ischaracterized by chronic inflammation of the skin. This inflammationhelps drive the formation of red, itchy skin plaques that are oftenpainful and disfiguring. TNF-α plays a critical role in their formationand continued existence because it induces synthesis if IL-1 and IL-8,the triggers of inflammation. TNF-α concentrations are higher inpsoriatic lesions than in unaffected skin of psoriatic patients and tendto decline with clearing of the lesions after effective therapy (Mussiet al., J. Biol. Regul. Homeost. Agents 11:115-118 [1997]). TNF-α alsopromotes keratinocyte proliferation and angiogenesis (Asadullah et al.,Drugs today 35:913-924 [1999]), and thus inhibiting this cytokine shouldhalt the disease at multiple stages.

Traditionally, first-line treatment of moderate psoriasis has consistedof topical agents because they are often less invasive than systemictherapy, with a low incidence of the most serious side effects, such asrenal or hepatic failure (Kincaid (2005) Drug discovery today 10:884).Some agents such as: antithyroid thioureylenes, propylthiouracil andmethimazole, are effective in the treatment of patients with psoriasiswith a significant number of patients showing clearing or near clearingof their lesions after a several weeks of treatment. Systemic treatmentwith the new anti-TNFα biologics: Enbrel, Remicade, and Humira, has beenapproved for moderate to severe chronic plaque psoriasis. Patientstreated with such agents very often show marked improvement in theirdisease with major clearing in several instances (Chaudhari et al.,Lancet 357:1842-1847 [2001]; Leonardi et al., N. Engl. J. Med.349:2014-2022 [2003]). But present day therapy of the disease is notparticularly satisfactory and the many therapies currently in use areassociated with significant cumulative toxicity (Gottlieb et al., J AmAcad Dermatol. 48:829-835 [2003]). Risks associated with TNFα blockingagents include serious infections, malignancies, anaphylaxis, hepatitisB reactivation, demyelinating disease, cytopenias, heart failure, andlupus-like syndrome. In addition, there is observed loss of clinicalbenefit after the drugs are stopped, and a small proportion of patientsdevelop antibodies to the biological agents which is likely to limittheir efficacy with repeated use.

Proteins such as the engineered modified variant BBPIs which are muchsmaller in size and thus presumed to be much less immunogenic shouldsignificantly reduce concerns about development of neutralizingantibodies in patients. BBPI molecules, because of their reducedmolecular weight in comparison to antibodies or proteins fusions, willlikely be more amenable to delivery via a topical route, or via lessinvasive systemic administrations like subcutaneous injections orneedleless delivery methods.

The invention provides personal care compositions comprising a modifiedvariant TNF-BBPI for use in skin and/or hair care. In some embodiments,the personal care composition comprising a TNF-BBPI is used forpromoting hair growth and/or improving the condition of the skin of asubject suffering from a dermatological inflammatory disorder. In someembodiments, the inflammatory skin disorder is chosen from dermatitis,eczema, psoriasis, acne, rosacea and hives. In some embodiments, thedermatological inflammatory disorder is psoriasis. Thus, the inventionprovides for personal skin care and/or hair care compositions. TheTNF-BBPIs comprised in the personal care compositions of the inventionare modified variant BBPIs in which the equivalent chymotrypsin loop ofthe precursor scaffold of the TNF-5-BBPI has been replaced by a TNFvariant peptide, and which further comprise at least one amino acidsubstitution as described in sections 5.4.1 and 5.4.2. In the presentinvention, binding of the modified variant TNF-BBPI to TNFα diverts TNFαfrom interacting with its cognate receptor and inhibits the TNFα-inducedinflammation of the scalp and preventing hair loss. In some embodiments,binding of the modified variant TNF-BBPI to TNFα diminishes theinflammation of the skin and improves the condition of the skin in asubject suffering from a dermatological inflammatory disorder recitedherein e.g. psoriasis. However, it is not intended that the presentinvention be limited to any particular mechanism.

In some embodiments, the TNF variant peptide comprised in the TNF-BBPIcomposition is chosen from RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQID NO:475), DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583),ATFPNQS (SEQ ID NO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ IDNO:586), AWHSPSV (SEQ ID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQID NO:589), GQTHFHV (SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH(SEQ ID NO:592), SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594). Inother embodiments, the TNF variant peptide is chosen from SEQ IDNOS:483, 484 AND 485.

The scaffold in which the variant TNF peptide is introduced to replacethe equivalent chymotrypsin loop is chosen from the scaffolds of thesoybean inhibitor from Glycine max (BBI; SEQ ID NO:13) or the mature andtruncated form thereof (SEQ ID NO:185), the inhibitor from Dolichosbiflorus (BBdb; SEQ ID NO:449), the soybean inhibitor D-II from Glycinemax (BBsb3; SEQ ID NO:450), the inhibitor from Torresea (Amburana)cearensis (BBtc; SEQ ID NO:451), the BBI-AV scaffold of (SEQ ID NO:186),the BBIt-AV scaffold of (SEQ ID NO:187), the BBdb-AV scaffold of (SEQ IDNO:452), the BBsb3-AV scaffold of (SEQ ID NO:453), the BBtc-AV scaffoldof (SEQ ID NO:454), the BBIt-VEGK scaffold of (SEQ ID NO:640), theBBIt-VEGT scaffold of (SEQ ID NO:641) and the BBIt-VEGKD scaffold of (SEID NO:642). In addition, any wild-type BBPI precursor scaffolds, such asthose disclosed by Prakash et al. (supra), may be used to generatevariant BBPI scaffolds. In some embodiments, the scaffold of theVEGF-BBPI is that of SEQ ID NO:187. In some embodiments, the backbone ofthe modified variant TNF-BBPI comprises at least one amino acidsubstitution at least at one amino acid position chosen from positionsequivalent to 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55,and 65 of the variant BBI of SEQ ID NO:187, as recited in section 5.4.1.In other embodiments, the backbone of the modified variant TNF-BBPIcomprises a combination of amino acid substitutions chosen from acombination two, three, four, five, six, seven or eight amino acidsubstitutions as recited above in section 5.4.2.

In other embodiments, the backbone of the modified variant TNF-BBPIcomprises a combination of amino acid substitutions chosen from acombination of amino acid substitutions chosen from 13I-29P-50T-52A,13I-40K-50T-52A, 13I-25K-29P-52K, 13I-29P-40K-50T-52A,13I-25R-27A-29P-31A-50K-52T, 13I-25R-27A-29P-31A-40H-50K-52T,13I-25K-27A-29R-31E-40K-50Q-52Q, 13I-25K-27A-29R-31A-40H-50R-52L atequivalent positions in SEQ ID NO:187.

In some embodiments, the personal care compositions of the inventioncomprises a BBPI in which the equivalent chymotrypsin loop of theprecursor scaffold is replaced with a TNF variant peptide chosen fromSEQ ID NOS:474, 475 And 476,

wherein the scaffold is that of SEQ ID NO:187, and which comprises acombination of amino acid substitutions 13I-25R-27A-29P-31A-50K-52T.

In some embodiments, the personal care compositions comprise a TNF-BBPIchosen from the TNF-BBPIs of SEQ ID NOS:647, 648, and 649.

In some embodiments, the invention provides a personal care compositioncomprising a TNF-BBPI that binds to TNF. In alternative embodiments, thebinding of the TNF-BBPI to TNF blocks the downstream activity of TNF. Insome embodiments, the composition is capable of modulating inflammation.

In some embodiments, the personal care composition comprising a modifiedvariant BBPI-TNF is for use in skin care. In some embodiments, the skincare compositions are for use in improving the appearance and/orcondition of skin in a subject suffering from a skin disorder. Thus, insome embodiments, the personal care compositions for use in skin careinclude cosmetic compositions. In some embodiments, the skin disorder isan inflammatory skin disorder. In some embodiments, the inflammatoryskin disorder is psoriasis.

In one embodiment, the personal care composition comprising a TNF-BBPIis a skin care composition is chosen from skin creams, lotions, sprays,emulsions, colloidal suspensions, foams, aerosols, liquids, gels, sera,and solids. In another embodiment, the personal care composition is askin care composition selected from moisturizing body washes, bodywashes, antimicrobial cleansers, skin protective creams, body lotions,facial creams, moisturizing creams, facial cleansing emulsions, facialgels, facial sera, surfactant-based facial cleansers, facial exfoliatinggels, anti-acne treatments, facial toners, exfoliating creams, facialmasks, after shave balms, pre-shave balms, tanning compositions, skinlightening compositions, skin redness reduction compositions,sunscreens, depilatories, hair growth inhibitors, and radioprotectives.Radioprotectives are chosen from non-water-resistant sunscreens, verywater-resistant sunscreens, and water-in-silicone sunscreens.

In one embodiment, the personal care composition comprising a TNF-BBPIis a skin care composition comprising topically applied over-the-countercompositions, anti-fungal treatments, anti-acne treatments, skinprotectants, sunscreens, deodorants, and antiperspirants. In otherembodiments, the skin care composition is capable of lightening skintone, reducing redness, preventing skin tone darkening or preventingcolor development.

The present invention also provides personal care compositions that arecosmetic compositions. In some preferred embodiments, the cosmeticcompositions are selected from mascaras, pressed powder formulations,and foundations. In some preferred embodiments, the makeup compositionscomprise at least one pigment.

In some preferred embodiments, the makeup composition comprising atleast one pigment is a mascara selected from non-waterproof mascaras,waterproof mascaras, volumizing mascaras, lengthening mascaras, curlingmascaras, anhydrous waterproof mascaras, water-based mascaras, andeyelash or eyebrow treatments.

In yet additional embodiments, the makeup compositions are pressedpowder formulations selected from loose powders, blushes, eye shadows,and bronzing powders. In still further embodiments, the makeupcompositions are foundations selected from water-in-oil foundations,water-in-silicone foundations, oil-in-water foundations, anhydrousmakeup sticks, and cream-to-powder foundations.

In some embodiments, the personal skin care compositions comprise amodified variant TNF-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the TNF-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the TNF-BBPI is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

In other embodiments, the invention provides a personal care compositioncomprising a modified variant BBPIs that comprises a TNF variant peptide(TNF-BBPI) for use in hair care.

In some embodiments, the hair care compositions find use in improvingthe appearance and/or condition of scalp skin in patients suffering frompsoriasis.

In one embodiment, the hair care composition is selected from the groupconsisting of shampoos, conditioners, hair styling compositions, haircolorants, permanent wave formulations, creams, gels, mousses, sprays,emulsions, colloidal suspensions, liquids, foams, and solids.

In some embodiments, the hair care composition further comprises aradioprotective. As described for the personal skin care compositionsthe radioprotective is a sunscreen chosen from non-water-resistantsunscreens, very water-resistant sunscreens, and water-in-siliconesunscreens. In other embodiments, the radioprotective is a sunscreenchosen from non-water-resistant sunscreens, very water-resistantsunscreens, and water-in-silicone sunscreens.

In some embodiments, the personal hair care compositions comprise amodified variant VEGF-BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the VEGF-BBPI is present inan amount of about 0.0001% to about 5% by weight based on the totalweight of the composition. Also preferably, the VEGF-BBPI is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup, as well as other suitable make-up and cosmetic preparations. Insome embodiments, the carrier is preferably at least one selected fromthe group consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

Formulations

In additional preferred embodiments, the present invention providescosmetic and/or pharmaceutical compositions comprising at least onemodified variant BBPI, as set forth herein, and a physiologicallyacceptable carrier or excipient. Preferably, the compound is present inan amount of about 0.0001% to about 5% by weight, based on the totalweight of the composition. Also preferably, the compound is present inan amount of about 0.001% to about 0.5% by weight based on the totalweight of the composition. The composition may be in the form of anemulsified vehicle, such as a nutrient cream or lotion, a stabilized gelor dispersion system, a treatment serum, a liposomal delivery system, atopical pack or mask, a surfactant-based cleansing system such as ashampoo or body wash, an aerosolized or sprayed dispersion or emulsion,a hair or skin conditioner, styling aid, or a pigmented product such asmakeup. Preferably, the carrier is at least compound selected from thegroup consisting of water, propylene glycol, ethanol, propanol,glycerol, butylene glycol and polyethylene glycol.

It is contemplated that the present invention will find use in numerouspersonal care compositions. It is not intended that the presentinvention be limited to any particular format or type of composition.The following description provides exemplary, not limiting compositionscomprising the following invention.

Emulsions comprises one group of customary, commonly-used cosmetics. Theterm “emulsion” is generally used in reference to a heterogeneous systemof two liquids which are immiscible or miscible only to a limited extentwith one another, which are usually referred to as “phases.” One phaseis typically in the form of droplets (i.e., the “dispersed,”“discontinuous” or “internal” phase), while the other liquid forms acontinuous (i.e., “coherent” or “external”) phase. Less common forms ofapplication include multiple emulsions (i.e. those in which the dropletsof the dispersed [or discontinuous] phase, comprise for their partdroplets of a further dispersed phase, such as water/oil/water [W/O/W]emulsions and oil/water/oil [O/W/O] emulsions).

If the oil phase is finely distributed in the water phase, then this isan oil-in-water emulsion (O/W emulsion; e.g. milk). The basic characterof an O/W emulsion is determined by the water. These emulsions aregenerally less greasy on the skin, are rather matting, and absorb morerapidly into the skin than W/O (water-in-oil) emulsions.

Those of skill in the art are familiar with a large number of options offormulating stable W/O preparations for cosmetic and/or dermatologicaluses, including such formulations as creams and ointments, which arespreadable in the range from room temperature to skin temperature, aswell as lotions and milks, which are more flowable in this temperaturerange.

The stability of emulsions is dependent on their viscosity, inparticular on the viscosity of the external phase. An emulsion becomesunstable when the finely dispersed particles collect together to formrelatively large aggregates, and the droplets which are in contactcoalesce. This process is referred to as “coalescence.” The more viscousthe external phase of the emulsion, the slower the process ofcoalescence. Emulsions of “liquid” (=flowable) consistency are used invarious cosmetics (e.g., skin care lotions, cleansing lotions, facelotions, hand lotions, etc.). These compositions generally have aviscosity of from about 2000 mPa·s to about 10,000 mPa·s. The stabilityof flowable emulsions is deserving of particular attention since theconsiderably greater mobility of the particles promotes more rapidcoalescence.

It is known that liquid emulsions typically presently in use generallycomprise thickeners and are not stable toward relatively highelectrolyte concentrations. This is manifested in phase separation ofthe compositions. However, in some embodiments, it is desirable to usecertain electrolytes (e.g., water-soluble UV filters), in order to beable to utilize the other physical, chemical or physiological propertiesthereof. Although in many cases appropriate choice of the emulsifiersystem can provide remedies to a certain extent, other disadvantagesthen arise just as often.

For example, some disadvantages result due to the fact that emulsifiers,like ultimately any chemical substance, may trigger allergic reactionsor reactions based on oversensitivity (i.e., hypersensitivity) of theuser. The use of customary cosmetic emulsifiers is generally entirelywithout risk, although for some individuals, “hypoallergenic”compositions are necessary and/or preferred. Indeed, in someparticularly sensitive individuals, certain dermatoses are triggered byexposure to certain emulsifiers and simultaneous exposure to sunlight.Thus, as known to those in the art, in some compositions, particularemulsifiers are less preferred and/or are avoided.

It is possible to prepare emulsifier-free preparations. For example,some preparations have an oily phase which contains dispersed waterdroplets (i.e., it is similar to a W/O emulsion). Such systems aresometimes called “hydrodispersions” or “oleodispersions,” depending uponwhich is the disperse phase and which is the continuous phase.

For cosmetic technology, it is, however, neither necessary nor possibleto dispense with emulsifiers altogether, especially since there is acertain choice of particularly mild emulsifiers.

In some liposomal embodiments, the liposomes comprise water and one ormore ingredients capable of forming lipid bilayer vesicles that can holdone or more functional or active ingredient(s). Non-limiting examples ofingredients capable of forming lipid bilayer vesicles include:phospholipids, hydrogenated phosphatidylcholine, lecithin, cholesteroland sphingolipids. Preferred liposomes include, without limitation: a)lipoid liposome 0003 (composed of water and lecithin and glycerin); b)lipoid liposome 0300 (composed of water and phosphatidylcholine); c)lipoid liposome 0111 (composed of water, Ginkgo biloba leaf extract,denatured alcohol, hydrogenated lecithin and cholesterol); d)anti-irritant liposomes (composed of water, cola acuminata seed extract,bisabolol and phospholipids); e) vitamin C and E liposomes (composed ofwater, phospholipids, tocopheryl acetate and ascorbyl palmitate); f)firming liposomes (composed of water, butylene glycol, pyrus malus(Apple) fruit extract, phospholipids, tocopheryl acetate and carbomer);and g) moisturizing liposomes (composed of water, sodium PCA, tocopherylacetate, xanthan gum, arginine, lysine, glycine and proline).

In other embodiments, the personal care composition of the presentinvention further comprise at least one active ingredient in addition tothe scaffolds provide herein. There are numerous active ingredientsknown to those of skill in the art that find use in the personal carecompositions of the present invention. Indeed, it is contemplated thatany suitable active ingredient or combination of suitable activeingredients will find use in the present invention (See e.g.,McCutcheon's Functional Materials, North American and InternationalEditions, published by MC Publishing Co. [2003]). For example, in someembodiments, the personal care compositions herein comprise a skin careactive ingredient at a level from about 0.0001% to about 20%, by weightof the composition. In another embodiment, the personal carecompositions comprise a skin care active ingredient from about 0.001% toabout 0.5%, by weight of the composition. In yet another embodiment, thepersonal care compositions comprise a skin care active ingredient fromabout 0.01% to about 2%, by weight of the composition.

Non-limiting examples of functional or active ingredients that can bedelivered via liposomes include: vitamins and their derivatives,antioxidants, proteins and peptides, keratolytic agents, bioflavinoids,terpenoids, phytochemicals, and extracts of plant, marine or fermentedorigin. In a preferred embodiment, the composition further comprises askin care or hair care active. Active ingredients can include any of awide variety of ingredients such as are known in the art. (See e.g.,McCutcheon's Functional Materials, North American and InternationalEditions, (2003), published by MC Publishing Co.). Preferably, suchactives include but are not limited to antioxidants, such as tocopheryland ascorbyl derivatives, bioflavinoids, terpenoids, synthetics and thelike, vitamins and vitamin derivatives, hydroxyl- and polyhydroxy acidsand their derivatives, such as AHAs and BHAs and their reactionproducts, peptides and polypeptides and their derivatives, such asglycopeptides and lipophilized peptides, heat shock proteins andcytokines, enzymes and enzymes inhibitors and their derivatives, such asproteases, MMP inhibitors, catalases, glucose oxydase and superoxidedismutase, amino acids and their derivatives, bacterial, fungal andyeast fermentation products and their derivatives, including mushrooms,algae and seaweed and their derivatives, phytosterols and plant andplant part extracts and their derivatives and phospholipids and theirderivatives, anti-dandruff agents such as zinc pyrithione and deliverysystems containing them, as provided herein and/or known in the art.

In some preferred embodiments, the skin care active is selected from thegroup consisting of a Vitamin B3 component, panthenol, Vitamin E,Vitamin E acetate, retinol, retinyl propionate, retinyl palmitate,retinoic acid, Vitamin C, theobromine, alpha-hydroxyacid, farnesol,phytrantriol, salicylic acid, palmityl peptapeptide-3 and mixturesthereof. In some preferred embodiments, the Vitamin B3 component isniacinamide. In some embodiments, the compositions provided hereincomprise a skin care active at a level from about 0.0001% to about 20%,preferably from about 0.001% to about 0.5%, more preferably from about0.01% to about 1%, by weight.

Exemplary derivatives of the foregoing vitamin B₃ compounds includenicotinic acid esters, including non-vasodilating esters of nicotinicacid, nicotinyl amino acids, nicotinyl alcohol esters of carboxylicacids, nicotinic acid N-oxide and niacinamide N-oxide. Suitable estersof nicotinic acid include nicotinic acid esters of C₁-C₂₂, preferablyC₁-C₁₆, more preferably C₁-C₆ alcohols. In these embodiments, thealcohols are suitably straight-chain or branched chain, cyclic oracyclic, saturated or unsaturated (including aromatic), and substitutedor unsubstituted. The esters are preferably non-vasodilating.

Non-vasodilating esters of nicotinic acid include tocopherol nicotinateand inositol hexanicotinate; tocopherol nicotinate are preferred. A morecomplete description of vitamin B₃ compounds is provided in WO 98/22085.Preferred vitamin B₃ compounds include niacinamide and tocopherolnicotinate.

In additional embodiments, the skin care active comprises at least oneretinoid. The retinoid is preferably retinol, retinol esters (e.g.,C₂-C₂₂ alkyl esters of retinol, including retinyl palmitate, retinylacetate, retinyl proprionate), retinal, and/or retinoic acid (includingall-trans retinoic acid and/or 13-cis-retinoic acid), more preferablyretinoids other than retinoic acid. These compounds are well known inthe art and are commercially available from a number of sources (e.g.,Sigma and Boehringer Mannheim). Preferred retinoids include retinol,retinyl palmitate, retinyl acetate, retinyl proprionate, retinal,retinoic acid and combinations thereof. More preferred are retinol,retinoic propionate, retinoic acid and retinyl palmitate. In someembodiments, the retinoid is included as a substantially pure material,while in other embodiments, it is provided as an extract obtained bysuitable physical and/or chemical isolation from natural (e.g., plant)sources. When a retinoid is included in the compositions herein, itpreferably comprises from about 0.005% to about 2%, preferably fromabout 0.01% to about 1% retinoid. Retinol is preferably used in anamount of from about 0.01% to about 0.15%; retinol esters are preferablyused in an amount of from about 0.01% to about 2% (e.g., about 1%).

In still further embodiments of the present invention, antioxidants areincorporated in the personal care compositions. It is contemplated thatany suitable antioxidants will find use in the personal carecompositions of the present invention. Suitable antioxidants include,but are not limited to amino acids (e.g., glycine, histidine, tyrosine,and tryptophan) and derivatives thereof, imidazoles (e.g. urocanic acid)and derivatives thereof, peptides (e.g., D,L-carnosine, D-carnosine, andL-carnosine) and derivatives thereof (e.g., anserine), carotenoids,carotenes (e.g., α-carotene, β-carotene, and γ-lycopene) and derivativesthereof, chlorogenic acid and derivatives thereof, aurothioglucose,propylthiouracil and other thiols (e.g., thioredoxin, glutathione,cysteine, cystine, cystamine and the glycosyl, N-acetyl, methyl, ethyl,propyl, amyl, butyl and lauryl, palmitoyl, oleyl, g-linoleyl,cholesteryl and glyceryl esters thereof) and salts thereof, dilaurylthiodipropionate, distearyl thiodipropionate, thiodipropionic acid andderivatives thereof (e.g., esters, ethers, peptides, lipids,nucleotides, nucleosides and salts), and sulfoximine compounds (e.g.buthionine sulfoximines, homocysteine sulfoximine, buthionine sulfones,penta-, hexa-, and heptathionine sulfoximine) in very small tolerateddoses (e.g., typically pmol to mmol/kg), chelating agents (e.g.,α-hydroxy fatty acids, palmitic acid, phytic acid, and lactoferrin),α-hydroxy acids (e.g. citric acid, lactic acid, and malic acid), humicacid, bile acid, bile extracts, bilirubin, biliverdin, EDTA, EGTA andderivatives thereof, unsaturated fatty acids and derivatives thereof(e.g., linolenic acids, linoleic acid, oleic acid), folic acid andderivatives thereof, furfurylidenesorbitol and derivatives thereof,ubiquinone and ubiquinol and derivatives thereof, vitamin C andderivatives thereof (e.g., sodium ascorbyl phosphate, ascorbylpalmitate, Mg ascorbyl phosphate, and ascorbyl acetate), tocopherols andderivatives (e.g., vitamin E acetate), coniferyl benzoate of benzoinresin, ferulic acid, furfurylideneglucitol, carnosine,butylhydroxytoluene, butylhydroxyanisole, nordihydroguaiacic acid,nordihydroguaiaretic acid, trihydroxybutyrophenone, uric acid andderivatives thereof, mannose and derivatives thereof, zinc andderivatives thereof (e.g., ZnO, ZnSO4), selenium and derivatives thereof(e.g., selenomethionine), stilbenes and derivatives thereof (e.g.,stilbene oxide, trans-stilbene oxide) and the derivatives thereof (e.g.,salts, esters, ethers, sugars, nucleotides, nucleosides, peptides andlipids) of said active ingredients which are suitable for the intendeduse of the particular embodiment(s) of the present invention.

In some embodiments, the concentration of one or more antioxidant in thecompositions of the present invention is preferably from about 0.001 toabout 30% by weight, particularly preferably from about 0.05 to about20% by weight, and more preferably from about 1 to about 10% by weight,based on the total weight of the preparation. In additional embodiments,in which vitamin E and/or its derivatives are utilized asanti-oxidant(s), the preferred range is from about 0.001 to about 10% byweight, based on the total weight of the formulation. However, it is notintended that the present invention be limited to any specificantioxidant concentration(s), as various concentrations will find use inthe various embodiments of the present invention.

In yet some additional embodiments, the active ingredient(s) is/arecatechins, bile esters of catechins, and/or aqueous or organic extractsfrom plants or sections of plants which have a content of catechins orbile esters of catechins (e.g., the leaves of the Theaceae plant family,in particular of the species Camellia sinensis [green tea]). Theirtypical ingredients (e.g., polyphenols or catechins, caffeine, vitamins,sugars, minerals, aminoacids, lipids) find particular use in someembodiments of the present invention.

In some embodiments, catechins find use in the present invention.Catechins are a group of compounds which are regarded as hydrogenatedflavones or anthocyanidines, and are derivatives of “catechin”(catechol, 3,3′,4′,5,7-flavanpentol,2-(3,4-dihydroxyphenyl)chroman-3,5,7-triol). Epicatechin((2R,3R)-3,3′,4′,5,7-flavanpentol) is also an active ingredient thatfinds use in some embodiments of the present invention.

In yet additional embodiments, plant extracts with a content ofcatechin, in particular extracts of green tea (e.g., extracts fromleaves of the plants of the genus Camellia, in particular those used fortea, such as C. sinenis, C. assamica, C. taliensis. and C. irrawadiensisand hybrids of these species with other species, such as C. japonica)find use in some personal care compositions of the present invention.

In some further embodiments, preferred active ingredients includepolyphenols and catechins from the group (−)-catechin, (+)-catechin,(−)-catechin gallate, (−)-gallocatechin gallate, (+)-epicatechin,(−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, and(−)-epigallocatechin gallate.

In some additional embodiments of the compositions of the presentinvention flavone and its derivatives (also often collectively called“flavones”) find used. These compounds have the following basicstructure (substitution positions are shown):

Some of the more important flavones which find use in some personal carecompositions of the present invention are provided below. However, it isnot intended that the present invention be limited to any particularflavone.

FLAVONES OH Substitution Positions 3 5 7 8 2′ 3′ 4′ 5′ (a) Flavone − − −− − − − − Flavonol + − − − − − − − Chrysin − + + − − − − −Galangin + + + − − − − − Apigenin − + + − − − + − Fisetin + − + − − + +− Luteolin − + + − − + + − Kaempferol + + + − − − + − Quercetin + + + −− + + − Morin + + + − + − + − Robinetin + − + − − + + +Gossypetin + + + + − + + − Myricetin + + + − − + + + (b) Flavone − − − −− − − − Flavonol + − − − − − − − Chrysin − + + − − − − − Galangin + + +− − − − − Apigenin − + + − − − + − Fisetin + − + − − + + − Luteolin− + + − − + + − Kaempferol + + + − − − + − Quercetin + + + − − + + −Morin + + + − + − + − Robinetin + − + − − + + + Gossypetin + + + + − + +− Myricetin + + + − − + + +

In nature, flavones are usually present in glycosylated form.

In some further embodiments, the personal care compositions of thepresent invention comprise at least one flavonoids having genericstructural formula:

where Z₁ to Z₇, independently of one another, are chosen from the groupconsisting of H, OH, alkoxy and hydroxyalkoxy, where the alkoxy andhydroxyalkoxy groups can be branched or unbranched and have 1 to 18carbon atoms, and where Gly is chosen from the group of mono- andoligoglycoside radicals.

In some alternative embodiments, the personal care compositions of thepresent invention comprise at least one flavonoids having the genericstructural formula:

where Z₁ to Z₆, independently of one another, are chosen from the groupconsisting of H, OH, alkoxy and hydroxyalkoxy, where the alkoxy andhydroxyalkoxy groups may be branched or unbranched and have 1 to 18carbon atoms, where Gly is chosen from the group mono and oligoglycosideradicals.

In some preferred embodiments, the composition has the genericstructural formula

where Gly₁, Gly₂ and Gly₃, independently of one another, aremonoglycoside radicals. Gly₂ and Gly₃ may also, individually ortogether, represent saturations by hydrogen atoms. In some preferredembodiments, Gly₁, Gly₂ and Gly₃, independently of one another, areselected from the group of hexosyl radicals, in particular the rhamnosylradicals and glucosyl radicals. However, hexosyl radicals, for exampleallosyl, altrosyl, galactosyl, gulosyl, idosyl, mannosyl and talosyl,also find use in some embodiments of the present invention. In yetadditional embodiments, pentosyl radicals find use in some personal carecompositions of the present invention.

In some embodiments, Z₁ to Z₅ are, independently of one another,advantageously chosen from the group consisting of H, OH, methoxy,ethoxy and 2-hydroxyethoxy, and the flavone glycosides have thestructure:

In some embodiments, the flavone glycosides provided in some of thepersonal care compositions of the present invention have the followingstructure:

where Gly₁, Gly₂ and Gly₃, independently of one another, aremonoglycoside radicals. Gly₂ and Gly₃ can also, individually ortogether, represent saturations by hydrogen atoms. In alternativeembodiments, Gly₁, Gly₂ and Gly₃, independently of one another, areselected from the group of hexosyl radicals, in particular of rhamnosylradicals and glucosyl radicals. However, other hexosyl radicals, forexample allosyl, altrosyl, galactosyl, gulosyl, idosyl, mannosyl andtalosyl, find use in some embodiments of the present invention. Inaddition, in some embodiments, pentosyl radicals find use in the presentinvention. In some preferred embodiments, the personal care compositionsof the present invention comprise one or more flavone glucoside selectedfrom the group consisting of a-glucosylrutin, a-glucosylmyricetin,a-glucosylisoquercitrin, a-glucosylisoquercetin anda-glucosylquercitrin. In some particularly preferred embodiments, theflavone glucoside is a-glucosylrutin.

In yet some additional embodiments, the personal care compositions ofthe present invention comprise at least one naringin (e.g., aurantin,naringenin-7-rhamno-glucoside), hesperidin3′,5,7-trihydroxy-4′-methoxyflavanone-7-rutinoside, hesperidoside,hesperetin-7-O-rutinoside), rutin(3,3′,4′,5,7-pentahydroxyflavone-3-rutinoside, quercetin-3-rutinoside,sophorin, birutan, rutabion, taurutin, phytomelin, melin), troxerutin(3,5-dihydroxy-3′,4′,7-tris(2-hydroxyethoxy)flavone-3-(6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside)),monoxerutin(3,3′,4′,5-tetrahydroxy-7-(2-hydroxyethoxy)flavone-3-(6-O-(6-deoxy-a-L-mannopyranosyl)-b-D-glucopyranoside)), dihydrorobinetin (3,3′,4′,5′,7-pentahydroxyflavanone),taxifolin (3,3′,4′,5,7-pentahydroxyflavanone), eriodictyol-7-g lucoside(3′,4′,5,7-tetrahydroxyflavanone-7 glucoside), flavanomarein(3′,4′,7,8-tetrahydroxyflavanone-7 glucoside), and/or isoquercetin(3,3′,4′,5,7-pentahydroxyflavanone-3-(b-D-glucopyranoside). In some yetfurther embodiments, the active ingredient is selected from the groupconsisting of ubiquinones and plastoquinones. Ubiquinones arecharacterized by the structural formula:

Ubiquinones are the most widespread and the most investigatedbioquinones. Ubiquinones are referred to, depending on the number ofisoprene units linked in the side chain, as Q-1, Q-2, Q-3 etc., oraccording to the number of carbon atoms, as U-5, U-10, U-15 etc. Theypreferably arise with certain chain lengths (e.g. in some microorganismsand yeasts where n=6). In most mammals, including humans, Q10predominates. Coenzyme Q10 finds particular use in some embodiments ofthe present invention. Its structural formula is:

Plastoquinones have the general structural formula:

Plastoquinones differ in the number n of isoprene radicals and arereferred to accordingly (e.g. PQ-9 [n=9]). In addition, otherplastoquinones with varying substituents on the quinone ring exist insome embodiments.

In yet additional embodiments, the present invention providespreparations suitable for use as deodorants and/or antiperspirants. Itis contemplated that any of the active ingredients which commonly finduse in such preparations will also find use in various embodiments ofthe present invention. Additional components that are commonly used insuch preparations also find use in various embodiments of the presentinvention. Examples of such actives and inactive compounds include, butare not limited to odor maskers (e.g., perfumes), odor absorber (e.g.,phyllosilicates described in DE-P 40 09 347); as well asmontmorillonite, kaolinite, illite, beidellite, nontronite, saponite,hectorite, bentonite, smectite, and zinc salts of ricinoleic acid. Insome embodiments of the present invention, the range of activeingredients (i.e., one or more compounds) in such preparations ispreferably from about 0.001 to about 30% by weight; more preferably fromabout 0.05 to about 20% by weight; and most particularly in the range offrom about 1 to about 10% by weight, based on the total weight of thepreparation.

In some embodiments, the compositions of the present invention comprisesafe and effective amounts of a dermatologically acceptable carrier thatis suitable for topical application to the skin or hair within which theessential materials and optional other materials are incorporated toenable the essential materials and optional components to be deliveredto the skin or hair at an appropriate concentration. Thus, in someembodiments, the carrier acts as a diluent, dispersant, solvent or thelike for the essential components, ensuring that these components can beapplied and distributed evenly over the selected target at anappropriate concentration.

In further embodiments, an effective amount of one or more compoundsdescribed herein is also be included in compositions to be applied tokeratinous materials such as nails and hair, including but not limitedto those useful as hair spray compositions, hair styling compositions,hair shampooing and/or conditioning compositions, compositions appliedfor the purpose of hair growth regulation and compositions applied tothe hair and scalp for the purpose of treating seborrhoea, dermatitisand/or dandruff.

In yet additional embodiments, an effective amount of one or morecompounds described herein is included in compositions suitable fortopical application to the skin or hair. These compositions are providedin any suitable form, including but not limited to creams, lotions,gels, suspensions dispersions, microemulsions, nanodispersions,microspheres, hydrogels, emulsions (e.g., oil-in-water and water-in-oil,as well as multiple emulsions), and multilaminar gels and the like (Seee.g., Schlossman et al., The Chemistry and Manufacture of Cosmetics,[1998], incorporated by reference, herein). In some embodiments, thecompositions are formulated as aqueous or silicone compositions, whilein other embodiments they are formulated as emulsions of one or more oilphases in an aqueous continuous phase (or an aqueous phase in an oilphase).

The type of carrier utilized in the present invention depends on thetype of product form desired for the composition. The carrier can besolid, semi-solid or liquid. Suitable carriers include liquids,semi-solids (e.g., creams, lotions, gels, sticks, ointments, andpastes), sprays and mousses. Preferably the carrier is in the form of alotion, cream or a gel, more preferably one which has a sufficientthickness or yield point to prevent the particles from sedimenting. Insome embodiments, the carrier is inert, while in other embodiments itprovides dermatological benefits. In some embodiments, the carrier isapplied directly to the skin and/or hair, while in other embodiments, itis applied via a woven or non-woven wipe or cloth. In yet otherembodiments, it is in the form of a patch, mask or wrap. In stillfurther embodiments, it is aerosolized or otherwise sprayed or pumpedonto the skin and/or hair. The carrier chosen is physically andchemically compatible with the essential components described herein,and should not unduly impair stability, efficacy or other use benefitsassociated with the compositions of the present invention.

Preferred carriers contain a dermatologically acceptable, hydrophilicdiluent. Suitable hydrophilic diluents include water, organichydrophilic diluents such as C₂-C₁₀, preferably C₂-C₆, preferably, C₃-C₆monohydric alcohols and low molecular weight glycols and polyols,including propylene glycol, polyethylene glycol polypropylene glycol,glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters,1,2,6-hexametriol, pentylene glycol, hexylene glycol, sorbitol esters,ethoxylated ethers, propoxylated ethers, and combinations thereof. Thediluent is preferably liquid. Water is a preferred diluent. Thecomposition preferably comprises at least about 20% of the hydrophilicdiluent.

In some embodiments, suitable carriers also comprise an emulsioncomprising a hydrophilic phase, especially an aqueous phase, and ahydrophobic phase (e.g., a lipid, oil or oily material). As well knownto those skilled in the art, the hydrophilic phase is dispersed in thehydrophobic phase, or vice versa, to form respectively hydrophilic orhydrophobic dispersed and continuous phases, depending on thecomposition of ingredients. The term “dispersed phase” is a termwell-known to one skilled in the art of emulsion technology, used inreference to the phase which exists as small particles or droplets thatare suspended in and surrounded by a continuous phase. The dispersedphase is also known as the internal or discontinuous phase. The emulsionmay be or comprise (e.g., in a triple or other multi-phase emulsion) anoil-in-water emulsion or a water-in-oil emulsion such as awater-in-silicone emulsion. Oil-in-water emulsions typically comprisefrom about 1% to about 60% (preferably about 1% to about 30%) of thedispersed hydrophobic phase and from about 1% to about 99% (preferablyfrom about 10% to about 90%) of the continuous hydrophilic phase, whilewater-in-oil emulsions typically comprise from about 1% to about 98%(preferably from about 40% to about 90%) of the dispersed hydrophilicphase and from about 1% to about 50% (preferably about 1% to about 30%)of the continuous hydrophobic phase.

In further embodiments, the carrier also includes one or more componentsthat facilitate penetration through the upper stratum corneum barrier tothe lower levels of the skin. Examples of penetration enhancers include,but are not limited to, propylene glycol, azone, ethoxydiglycol,dimethyl isosorbide, urea, ethanol and dimethyl sulfoxide, as well asmicroemulsions, liposomes and nanoemulsions.

In some additional embodiments, the compositions of the presentinvention comprise humectants which are preferably present at a level offrom about 0.01% to about 20%, preferably from about 0.1% to about 15%and preferably from about 0.5% to about 10%. Preferred humectantsinclude, but are not limited to, compounds selected from polyhydricalcohols, sorbitol, glycerol, urea, betaine, D-panthenol, DL-panthenol,calcium pantothenate, royal jelly, panthetine, pantotheine, panthenylethyl ether, pangamic acid, pyridoxin, pantoyl lactose Vitamin Bcomplex, sodium pyrrolidone carboxylic acid, hexane-1,2,6,-triol,guanidine or its derivatives, and mixtures thereof.

Suitable polyhydric alcohols for use herein include, but are not limitedto polyalkylene glycols and preferably alkylene polyols and theirderivatives, including propylene glycol, dipropylene glycol,polypropylene glycol, polyethylene glycol and derivatives thereof,sorbitol, hydroxypropyl sorbitol, erythritol, threitol, pentaerythritol,xylitol, glucitol, mannitol, pentylene glycol, hexylene glycol, butyleneglycol (e.g., 1,3-butylene glycol), hexane triol (e.g.,1,2,6-hexanetriol), trimethylol propane, neopentyl glycol, glycerine,ethoxylated glycerine, propane-1,3 diol, propoxylated glycerine andmixtures thereof. The alkoxylated derivatives of any of the abovepolyhydric alcohols are also suitable for use herein. Preferredpolyhydric alcohols of the present invention are selected fromglycerine, butylene glycol, propylene glycol, pentylene glycol, hexyleneglycol, dipropylene glycol, polyethylene glycol, hexane triol,ethoxylated glycerine and propoxylated glycerine and mixtures thereof.

Suitable humectants useful herein are sodium 2-pyrrolidone-5-carboxylate(NaPCA), guanidine; glycolic acid and glycolate salts (e.g., ammoniumand quaternary alkyl ammonium); lactic acid and lactate salts (e.g.,ammonium and quaternary alkyl ammonium); aloe vera in any of its varietyof forms (e.g., aloe vera gel); hyaluronic acid and derivatives thereof(e.g., salt derivatives such as sodium hyaluronate); lactamidemonoethanolamine; acetamide monoethanolamine; urea; betaine, panthenoland derivatives thereof; and mixtures thereof.

In some embodiments, at least part (up to about 5% by weight ofcomposition) of a humectant is incorporated into the compositions of thepresent invention in the form of an admixture with a particulatecross-linked hydrophobic acrylate or methacrylate copolymer, itselfpreferably present in an amount of from about 0.1% to about 10%, whichcan be added either to the aqueous or disperse phase. This copolymer isparticularly valuable for reducing shine and controlling oil whilehelping to provide effective moisturization benefits and is described infurther detail in WO96/03964, incorporated herein by reference.

In some embodiments, the oil-in-water and water-in-oil compositions ofthe present invention comprise from about 0.05% to about 20%, preferablyfrom about 1% to about 15%, preferably from about 2% to about 10%,preferably from about 2% to about 5% of a dermatologically acceptableemollient. Emollients tend to lubricate the skin, increase thesmoothness and suppleness of the skin, prevent or relieve dryness of theskin and/or protect the skin. Emollients are typically water-immiscible,oily or waxy materials and emollients can confer aesthetic properties toa topical composition. A wide variety of suitable emollients are known(See e.g., Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol.1, pp. 32-43 [1972]; and WO 00/24372), and find use herein, containsnumerous examples of materials suitable as emollients. Additionalemollients include, but are not limited to the following:

i) Straight and branched chain hydrocarbons having from about 7 to about40 carbon atoms, such as mineral oils, dodecane, squalane, cholesterol,hydrogenated polyisobutylene, isohexadecane, isoeicosane,isooctahexacontane, isohexapentacontahectane, and the C₇-C₄₀isoparaffins, which are C₇-C₄₀ branched hydrocarbons. Suitable branchedchain hydrocarbons for use herein are selected fromisopentacontaoctactane, petrolatum and mixtures thereof;

ii) C₁-C₃₀ fatty acid esters of C₁-C₃₀ carboxylic acids, C₁₂₋₁₅ alkylbenzoates and of C₂-C₃₀ dicarboxylic acids, e.g. isononyl isononanoate,isostearyl neopentanoate, isodecyl octanoate, isodecyl isononanoate,tridecyl isononanoate, myristyl octanoate, octyl pelargonate, octylisononanoate, myristyl myristate, myristyl neopentanoate, myristyloctanoate, isopropyl myristate, myristyl propionate, isopropyl stearate,isopropyl isostearate, methyl isostearate, behenyl behenate, dioctylmaleate, diisopropyl adipate, and diisopropyl dilinoleate and mixturesthereof also find use in the present invention;

iii) C₁-C₃₀ mono- and poly-esters of sugars and related materials. Theseesters are derived from a sugar or polyol moiety and one or morecarboxylic acid moieties. Depending on the constituent acid and sugar,these esters can be in either liquid or solid form at room temperature.Examples include: glucose tetraoleate, the galactose tetraesters ofoleic acid, the sorbitol tetraoleate, sucrose tetraoleate, sucrosepentaoleate, sucrose hexaoleate, sucrose heptaoleate, sucroseoctaoleate, sorbitol hexaester. Other materials include cottonseed oilor soybean oil fatty acid esters of sucrose. Other examples of suchmaterials are described in WO 96/16636, incorporated by referenceherein;

iv) Vegetable oils and hydrogenated vegetable oils. Examples ofvegetable oils and hydrogenated vegetable oils include safflower oil,grapeseed oil, coconut oil, cottonseed oil, menhaden oil, palm kerneloil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, ricebran oil, pine oil, nut oil, sesame oil, sunflower seed oil, partiallyand fully hydrogenated oils from the foregoing sources and mixturesthereof;

v) Soluble or colloidally-soluble moisturizing agents. Examples includehyaluronic acid and chondroitin sulfate.

The term “lipid” is often used as a generic term to refer to fats, oils,waxes and the like. In addition, the terms “oil phase” and “lipid phase”are also used synonymously. However, oils and fats differ from oneanother in their polarity, which is difficult to define. It has beenproposed to adopt the interfacial tension toward water as a measure ofthe polarity index of an oil or of an oily phase. Thus, it iscontemplated that the interfacial tension be regarded as a suitablemeasure of the polarity of a given oil component. The “interfacialtension” is the force which acts on an imaginary line one meter inlength in the interface between two phases. In this measurement, thelower the interfacial tension between the oily phase and water, thegreater the polarity of the oily phase being analyzed. The physical unitfor this interfacial tension is conventionally calculated from theforce/length relationship and is usually expressed in mN/m (millinewtonsdivided by meters). It has a positive sign if it endeavours to reducethe interface. In the converse case, it has a negative sign. As usedherein, lipids are regarded as “polar,” if their interfacial tensiontoward water is less than 30 mN/m.

“Polar oils” include those from the group of lecithins and of fatty acidtriglycerides, namely the triglycerol esters of saturated and/orunsaturated, branched and/or unbranched alkane carboxylic acids having achain length of from 8 to 24, in particular 12 to 18, carbon atoms. Insome embodiments, the fatty acid triglycerides are chosen from the groupconsisting of synthetic, semi-synthetic and natural oils (e.g., oliveoil, sunflower oil, soya oil, groundnut oil, rapeseed oil, almond oil,palm oil, coconut oil, castor oil, wheatgerm oil, grapeseed oil, thistleoil, evening primrose oil, macadamia nut oil and the like). However, isit not intended that the present invention be limited to compositionsthat contain particular polar oils. Additional examples of polar oilsthat find use in the present invention include the group of esters ofsaturated and/or unsaturated, branched and/or unbranched alkanecarboxylic acids having a chain length of from 3 to 30 carbon atoms andsaturated and/or unsaturated, branched and/or unbranched alcohols havinga chain length of from 3 to 30 carbon atoms, and from the group ofesters of aromatic carboxylic acids and saturated and/or unsaturated,branched and/or unbranched alcohols having a chain length of from 3 to30 carbon atoms. In some embodiments, such ester oils are chosen fromthe group consisting of isopropyl myristate, isopropyl palmitate,isopropyl stearate, isopropyl oleate, n-butyl stearate, n-hexyl laurate,n-decyl oleate, isooctyl stearate, isononyl stearate, isononylisononanoate, 2-ethylhexyl palmitate, 2-ethylhexyl laurate, 2-hexyldecylstearate, 2-octyldodecyl palmitate, oleyl oleate, oleyl erucate, erucyloleate, erucyl erucate and synthetic, semi-synthetic and naturalmixtures of such esters (e.g., jojoba oil).

In addition, in some embodiments, the oily phase is chosen from thegroup consisting of dialkyl ethers, as well as saturated or unsaturated,and branched or unbranched alcohols. In some particularly preferredembodiments, the oily phase of the compositions of the preferredembodiments also contains C₁₂₋₁₅-alkyl benzoate, while in alternativeembodiments, the preferred embodiments contains only the latter. In yetadditional embodiments, the oil phase is chosen from the group ofGuerbet alcohols (i.e., the group of alcohols named after Marcel Guerbetwho first described their preparation). These alcohols are formedaccording to the equation:

by oxidation of an alcohol to an aldehyde, by aldol condensation of thealdehyde, elimination of water from the aldol and hydrogenation of theallyl aldehyde. Guerbet alcohols are liquid even at low temperatures andresult in virtually no skin irritations. Thus, they find use as fatting,superfatting and also refatting constituents in skincare and hair carecompositions. Indeed, the use of Guerbet alcohols is known in thecosmetic art. In these applications, the species are generallycharacterized as having the following structure:

In this structure, R₁ and R₂ are usually unbranched alkyl radicals. Insome preferred embodiments of the present invention the followingGuerbet alcohols in which R₁ is propyl, butyl, pentyl, hexyl, heptyl oroctyl and/or R₂ is hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,tridecyl or tetradecyl find use in the present invention. In additionalembodiments, preferred Guerbet alcohols include 2-butyloctanol with thefollowing chemical structure:

which is commercially available, for example, under the trade nameISOFOL® 12 (Condea Chemie GmbH), and 2-hexyldecanol with the followingchemical structure:

which is commercially available, for example, under the trade nameISOFOL® 16 (Condea Chemie GmbH).

In additional embodiments, mixtures of Guerbet alcohols find use incompositions of the present invention. For example, mixtures of2-butyloctanol and 2-hexyldecanol find use in some embodiments. Thetotal amount of Guerbet alcohols in the finished cosmetic ordermatological preparations is selected from the of range up to about25.0% by weight, preferably about 0.5 to about 15.0% by weight, based onthe total weight of the preparations. However, it is not intended thatthe present invention be limited to any particular concentration norrange of concentrations, as those of skill in the art know how toprepare compositions having suitable concentrations for the desiredcompositions and their use(s). In addition, it is contemplated that anymixtures of oil and/or wax components will find use in the presentinvention. For example, in some embodiments, waxes (e.g., cetylpalmitate) find use as the sole lipid component of the oil phase. Inadditional embodiments, nonpolar oils (e.g., those which are chosen fromthe group of branched and unbranched hydrocarbons and hydrocarbon waxes,in particular VASELINE® [i.e., petrolatum], paraffin oil, squalane andsqualene, polyolefins and hydrogenated polyisobutenes find use in thepresent invention. In some embodiments containing polyolefins,polydecenes are the preferred substances.

Fatty and/or wax components which find use in embodiments of the presentinvention include but are not limited to vegetable waxes, animal waxes,mineral waxes and petrochemical waxes. Examples which particularlypreferred waxes include candelilla wax, carnauba wax, japan wax, espartograss wax, cork wax, guaruma wax, rice germ oil wax, sugar cane wax,berry wax, ouricury wax, montan wax, jojoba wax, shea butter, beeswax,shellac wax, spermaceti, lanolin (wool wax), uropygial grease, ceresin,ozokerite (earth wax), paraffin waxes and microcrystalline waxes.

Additional fatty and/or wax components that find use in the presentinvention include chemically modified waxes and/or synthetic waxes(e.g., those commercially available under the trade names SYNCROWAX® HRC[glyceryl tribehenate] and SYNSCROWAX® AW 1C [C₁₈-C₃₆ fatty acid], whichare available from CRODA GmbH), and montan ester waxes, Sasol waxes,hydrogenated jojoba waxes, synthetic or modified beeswaxes (e.g.,dimethicone copolyol beeswax and/or C₃₀₋₅₀ alkyl beeswax), polyalkylenewaxes, polyethylene glycol waxes, as well as chemically modified fats(e.g., hydrogenated vegetable oils, such as hydrogenated castor oiland/or hydrogenated coconut fatty glycerides), triglycerides (e.g.,trihydroxystearin, fatty acids, fatty acid esters, and glycol esters,such as, C₂₀-C₄₀-alkyl stearate, C₂₀-C₄₀-alkylhydroxystearoyl stearateand/or glycol montanate). In further embodiments, the present inventioncomprises certain organosilicone compounds, which have similar physicalproperties to the specified fatty and/or wax components (e.g.,stearoxytrimethylsilane). In additional embodiments, the fatty and/orwax components are provided individually, while in still furtherembodiments, they are provided as a mixture. Indeed, it is intended thatany desired mixture of such oil and/or wax components will find use invarious embodiments of the present invention.

In some embodiments, the oily phase is selected from the groupconsisting of 2-ethylhexyl isostearate, octyldodecanol, isotridecylisononanoate, isoeicosane, 2-ethylhexyl cocoate, C₁₂-C₁₅-alkyl benzoate,caprylic/capric triglyceride, and dicaprylyl ether. In alternativeembodiments, mixtures of various oily phases are provided, including butnot limited to mixtures comprising one or more of octyldodecanol,caprylic/capric triglyceride, dicaprylyl ether, C₁₂-C₁₅-alkyl benzoate,2-ethylhexyl isostearate, isotridecyl isononanoate. The following tableprovides a list of lipids which find use alone or in combination withother lipids in various embodiments of the present invention. Thecorresponding interfacial tensions toward water are given in the lastcolumn. However, it is not intended that the present invention belimited to these specific components, as other components find use invarious embodiments of the present invention, including mixtures ofgreater or lesser polar components and the like.

LIPIDS Trade name Article II. INCI name (m/Nm) ISOFOL ® 14 T ButylDecanol + Hexyl Decanol + 27.6 Hexyl Octanol + Butyl Octanol ISOFOL ® 16Hexyl Decanol 24.3 EUTANOL ® G Octyldodecanol 24.8 CETIOL ® OEDicaprylyl Ether 22.1 MIGLYOL ® 812 Caprylic/Capric Triglyceride 21.3CEGESOFT ® C24 Octyl Palmitate 23.1 Isopropyl stearate IsopropylStearate 21.9 ESTOL ® 1540 EHC Octyl Octanoate 30.0 FINSOLV ® TN C₁₂-C₁₅Alkyl Benzoate 21.8 CETIOL ® SN Cetearyl Isonoanoate 28.6 DERMOFEEL ®BGC Butylene Glycol Dicaprylate/Dicapate 21.5 TRIVENT ® OCG Tricaprylin20.2 MOD Octyldodeceyl Myristate 22.1 COSMACOL ® ETI Di-C₁₂-C₁₃ AlkylTartrate 29.4 MIGLYCOL ® 829 Caprylic/Capric Diglyceryl Succinate 29.5PRISORINE ® 2036 Octyl Isostearate 29.7 TEGOSOFT ® SH Stearyl Heptanoate28.7 ABIL ® Wax 9840 Cetyl Dimethicone 25.1 CETIOL ® LCCoco-Caprylate/Caprate 24.8 IPP Isopropyl Palmitate 22.5 LUVITOL ® EHOCetearyl Octanoate 28.6 CETIOL ® 868 Octyl Stearate 28.4

In some embodiments, some or all of the oil phase of the preparationsare selected from the group consisting of cyclic and/or linear siliconeswhich are also often referred to as “silicone oils.” In someembodiments, these silicones or silicone oils are present as monomerswhich are generally characterized by structural elements as follows:

Silicones having two or more siloxyl units which find use in someembodiments of the present invention are generally characterized bystructural elements as follows:

where the silicon atoms may be substituted by identical or differentalkyl radicals and/or aryl radicals, which are represented in generalterms by the radicals R₁ to R₄, where the number of different radicalsis not necessarily limited to 4 and may assume values from 2 to 200,000.

Cyclic silicones to be used advantageously according to the inventionare generally characterized by the structural elements as follows:

where the silicon atoms may be substituted by identical or differentalkyl radicals and/or aryl radicals, which are represented here ingeneral terms by the radicals R₁ to R₄, where the number of differentradicals is not necessarily limited to 4. n can assume values of 3/2 to20. Fractional values for “n” take into consideration that unevennumbers of siloxyl groups may be present in the cycle.

In some embodiments, phenyltrimethicone is selected as silicone oil.Other silicone oils suitable for use in various embodiments of thepresent invention include, but are not limited to dimethicone,phenyldimethicone, cyclomethicone (octamethylcyclotetrasiloxane),hexamethylcyclotrisiloxane, polydimethylsiloxane,poly(methylphenylsiloxane), cetyldimethicone, and behenoxydimethicone.In alternative embodiments, mixtures of these compounds find use in thepresent invention, including but not limited to mixtures ofcyclomethicone and isotridecyl isononanoate, and mixtures ofcyclomethicone and 2-ethylhexyl isostearate. It yet additionalembodiments, silicone oils of similar constitution, such as thecompounds referred to above whose organic side chains have beenderivatized (e.g., polyethoxylated and/or polypropoxylated) find use inthe present invention. These include, but are not limited to suchcompounds as polysiloxane-polyalkyl-polyether copolymers such ascetyldimethicone copolyol (i.e., cetyldimethicone copolyol (and)polyglyceryl-4 isostearate (and) hexyl laurate). Indeed, it is notintended that the present invention be limited to any specific siliconeoil nor mixture of silicone oils, as various oils find use in variousembodiments of the present invention.

In additional embodiments, water in oil (W/O) emulsions find use in thepresent invention. In some embodiments, W/O emulsifiers are used with orwithout additional co-emulsifiers. In still further embodiments, W/Oemulsions of the present further comprise one or more emulsifiers,including, but not limited to one or more of the following compounds:lecithin, lanolin, microcrystalline wax (Cera microcristallina) in amixture with paraffin oil (Paraffinum liquidum), ozokerite, hydrogenatedcastor oil, polyglyceryl-3 oleate, wool wax acid mixtures, wool waxalcohol mixtures, pentaerythrithyl isostearate, polyglyceryl-3diisostearate, beeswax (Cera alba) and stearic acid, sodiumdihydroxycetylphosphate in a mixture with isopropyl hydroxycetyl ether,methylglucose dioleate, methylglucose dioleate in a mixture withhydroxystearate and beeswax, mineral oil in a mixture with petrolatumand ozokerite and glyceryl oleate and lanolin alcohol, petrolatum in amixture with ozokerite and hydrogenated castor oil and glycerylisostearate and polyglyceyl-3 oleate, PEG-7 hydrogenated castor oil,ozokerite and hydrogenated castor oil, polyglyceryl-4 isostearate,polyglyceryl-4 isostearate in a mixture with cetyldimethicone copolyoland hexyl laurate, laurylmethicone copolyol, cetyldimethicone copolyol,acrylate/C₁₀-C₃₀-alkyl acrylate crosspolymer, Poloxamer 101,polyglyceryl-2 dipolyhydroxystearate, polyglyceryl-3 diisostearate,polyglyceryl-4 dipolyhydroxystearate, PEG-30 dipolyhydroxystearate,diisostearoyl polyglyceryl-3 diisostearate, polyglyceryl-2dipolyhydroxystearate, polyglyceryl-3 dipolyhydroxystearate,polyglyceryl-4 dipolyhydroxystearate, polyglyceryl-3 dioleate.

In yet additional embodiments of the present invention, W/O emulsions ofthe present invention comprise one or more coemulsifiers, including, butnot limited to the following:

glyceryl stearate in a mixture with ceteareth-20, ceteareth-25,ceteareth-6 in a mixture with stearyl alcohol, cetylstearyl alcohol in amixture with PEG-40 castor oil and sodium cetylstearyl sulfate,triceteareth-4 phosphate, sodium cetylstearyl sulfate, lecithintrilaureth-4 phosphate, laureth-4 phosphate, stearic acid, propyleneglycol stearate SE, PEG-25 hydrogenated castor oil, PEG-54 hydrogenatedcastor oil, PEG-6 caprylic/capric glycerides, glyceryl oleate in amixture with propylene glycol, ceteth-2, ceteth-20, polysorbate 60,glyceryl stearate in a mixture with PEG-100 stearate, laureth-4,ceteareth-3, isostearyl glyceryl ether, cetylstearyl alcohol in amixture with sodium cetylstearyl sulfate, laureth-23, steareth-2,glyceryl stearate in a mixture with PEG-30 stearate, PEG-40 stearate,glycol distearate, PEG-22 dodecyl glycol copolymer, polyglyceryl-2 PEG-4stearate, ceteareth-20, methylglucose sesquistearate, steareth-10,PEG-20 stearate, steareth-2 in a mixture with PEG-8 distearate,steareth-21, steareth-20, isosteareth-20, PEG-45/dodecyl glycolcopolymer, methoxy-PEG-22/dodecyl glycol copolymer, PEG-20 glycerylstearate, PEG-8 beeswax, polyglyceryl-2 laurate, isostearyl diglycerylsuccinate, stearamidopropyl PG dimonium chloride phosphate, glycerylstearate SE, ceteth-20, triethyl citrate, PEG-20 methylglucosesesquistearate, ceteareth-12, glyceryl stearate citrate, cetylphosphate, triceteareth-4 phosphate, trilaureth-4 phosphate,polyglyceryl methylglucose distearate, potassium cetyl phosphate,isosteareth-10, polyglyceryl-2 sesquiisostearate, ceteth-10, oleth-20,isoceteth-20, glyceryl stearate in a mixture with ceteareth-20,ceteareth-12, cetylstearyl alcohol and cetyl palmitate, cetylstearylalcohol in a mixture with PEG-20 stearate, PEG-30 stearate, PEG-40stearate, and PEG-100 stearate.

In yet additional embodiments in which the oil phase of the preparationsconsists at least partially of silicone oils, silicone emulsifiers finduse. In some embodiments, the silicone emulsifiers are selected from thegroup of interface-active substances, alkylmethicone copolyols, and/oralkyl dimethicone copolyols, particularly from the group of compoundscharacterized by the following chemical structure:

in which X and Y, independently of one another, are chosen from thegroup H and the branched and unbranched alkyl groups, acyl groups andalkoxy groups having 1 to 24 carbon atoms, p is a number from 0 to 200,q is a number from 1 to 40, and r is a number from 1 to 100. Someexamples of silicone emulsifiers which find use in the present inventioninclude, but are not limited to dimethicone copolyols (e.g., ABIL® B8842, ABIL® B 8843, ABIL® B 8847, ABIL® B 8851, ABIL® B 8852, ABIL® B8863, ABIL® B 8873, and ABIL® B 88183, all of which are commerciallyavailable from Th. Goldschmidt AG). An additional example of aninterface-active substances which finds use in the present inventionincludes cetyldimethicone copolyol (ABIL® EM 90), as well ascyclomethiconedimethicone copolyol (ABIL® EM 97), both of which arecommercially available from Th. Goldschmidt AG. An additional emulsifierwhich has proven useful in various compositions that finds use inembodiments of the present invention is laurylmethicone copolyol (DowCorning® 5200 Formulation Aid), which is commercially available from DowCorning Ltd.

In preferred embodiments of the present invention, the total amount ofemulsifiers used in the personal care compositions (e.g., cosmetic orskin care preparations) are present in the range from about 0.1 to about10.0% by weight, preferably about 0.5 to about 5.0% by weight, based onthe total weight of the preparations. However, it is not intended thatthe present invention be limited to any specific concentration ofemulsifier and/or co-emulsifier, as various embodiments of the presentinvention have different preferred concentrations and/or concentrationranges.

In some embodiments, the present invention provides emulsions in variousforms, including skin protection creams, skin lotions, cosmetic milks,sunscreen creams, and sun protection milks. In some preferredembodiments, these compositions comprise fats, oils, waxes, and/or otherfatty substances, as well as water, and one or more emulsifiers as arecustomarily used for such a type of formulation.

In addition to the liquid and somewhat more solid emulsions of thecosmetic cleansing lotions and/or cleansing creams of the presentinvention, the present invention also provides sprayable cleansingpreparations (“cleansing sprays”), which are used, for example, forremoving make-up or as mild washing lotion. In addition, these cleansingsprays find use in applications for treatment of blemished skin. Thesecleansing preparations also find use as “rinse-off preparations” (i.e.,products which are rinsed off the skin following application).

In addition to the above constituents, various embodiments of thepresent invention include additional components, such as auxiliaries andadditives, including but not limited to bodying agents, fillers,perfume, dyes, emulsifiers, additional active ingredients (e.g.,vitamins and proteins), light protection agents, stabilizers, insectrepellents, alcohol, self-tanning substances, water, salts,antimicrobials, proteases, and/or keratinase, etc. Indeed, it is notintended that the present invention be limited to any particularcomponents, as long as the active component comprising a scaffold and apeptide is included. It is further contemplated that the presentinvention will find use in numerous and various medicinal preparations.

In some embodiments, the compositions of the present invention containan emulsifier and/or surfactant, generally to help disperse and suspendthe disperse phase within the continuous aqueous phase. A surfactant mayalso be useful if the product is intended for skin or hair cleansing.For convenience hereinafter, “emulsifiers” are encompassed by the term“surfactants.” Thus, as used herein, the term “surfactant(s)” refers tosurface active agents, whether used as emulsifiers or for othersurfactant purposes such as skin cleansing. Known, includingconventional surfactants find use in the present invention, providedthat the selected agent is chemically and physically compatible withessential components of the composition and provides the desiredcharacteristics (See e.g., WO 00/24372). Suitable surfactants includenon-silicone derived materials, silicone-derived materials, and mixturesthereof.

In further embodiments, the compositions of the present inventioncomprise preferably from about 0.05% to about 30%, more preferably fromabout 0.5% to 15%, and most preferably from about 1% to 10% of asurfactant or mixture of surfactants. The exact surfactant or surfactantmixture chosen depends upon the pH of the composition, the othercomponents present and the desired final product aesthetics.

Among the nonionic surfactants that are useful herein are those that canbe broadly defined as condensation products of long chain alcohols(e.g., C₈₋₃₀ alcohols), with sugar or starch polymers (e.g.,glycosides). Other useful nonionic surfactants include the condensationproducts of alkylene oxides with fatty acids (i.e., alkylene oxideesters of fatty acids). These materials have the general formulaRCO(X)_(n)OH wherein R is a C₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— (i.e.,derived from ethylene glycol or oxide) or —OCH₂CHCH₃— (i.e., derivedfrom propylene glycol or oxide) and n is an integer from about 6 toabout 200. Other nonionic surfactants are the condensation products ofalkylene oxides with 2 moles of fatty acids (i.e., alkylene oxidediesters of fatty acids). These materials have the general formulaRCO(X)_(n)OOCR wherein R is a C₁₀₋₃₀ alkyl group, X is —OCH₂CH₂— (i.e.,derived from ethylene glycol or oxide) or —OCH₂CHCH₃— (i.e., derivedfrom propylene glycol or oxide) and n is an integer from about 6 toabout 100. In some embodiments, an emulsifier for use herein ispreferably a fatty acid ester blend based on a mixture of sorbitan fattyacid ester and sucrose fatty acid ester, especially a blend of sorbitanstearate and sucrose cocoate. Further suitable examples include amixture of cetearyl alcohols and cetearyl glucosides. However, it is notintended that the present invention be limited to any particularemulsifier, as various suitable emulsifiers are known in the art.

In additional embodiments, the hydrophilic surfactants useful hereinalternatively or additionally include any of a wide variety of cationic,anionic, zwitterionic, and amphoteric surfactants such as are known inthe art (See, e.g., McCutcheon's, Emulsifiers and Detergents, NorthAmerican and International Editions, MC Publishing Co. [2003]; U.S. Pat.Nos. 5,011,681 4,421,769; and 3,755,560).

In some additional embodiments, interface- and/or surface-active agentsare included in some personal care compositions of the presentinvention, including but not limited to cationic emulsifiers (e.g.,quaternary surfactants).

Quaternary surfactants that contain at least one N atom which iscovalently bonded to 4 alkyl or aryl groups. This leads, irrespective ofthe pH, to a positive charge. Alkylbetain, alkylamidopropylbetain andalkylamidopropylhydroxysultaine are examples of quaternary surfactantsthat find use in some embodiments of the present invention.

The cationic surfactants provided in some embodiments of the presentinvention also include, but are not limited to quaternary ammoniumcompounds, in particular benzyltrialkylammonium chlorides or bromides(e.g., benzyldimethylstearylammonium chloride), alkyltrialkylammoniumsalts (e.g., cetyltrimethylammonium chloride or bromide),alkyldimethylhydroxyethylammonium chlorides or bromides,dialkyldimethylammonium chlorides or bromides,alkylamidoethyltrimethylammonium ether sulfates, alkylpyridinium salts(e.g., lauryl- or cetylpyrimidinium chloride), imidazoline derivatives,and compounds with a cationic character, such as amine oxides (e.g.,alkyldimethylamine oxides or alkylaminoethyldimethylamine oxides). Insome preferred embodiments, cetyltrimethylammonium salts find use insome personal care compositions of the present invention.

In yet additional embodiments, cationic polymers (e.g., JAGUAR® C 162[hydroxypropyl guar hydroxypropyltrimonium chloride]), modifiedmagnesium aluminum silicates (e.g., quaternium-18-hectorite, which iscommercially available (e.g., BENTONE® 38; Rheox), and/or stearalkoniumhectorite, which is commercially available (e.g., SOFTISAN® gel; HülsAG) find use in some personal care compositions of the presentinvention. However, it is not intended that the present invention belimited to any particular cationic polymer.

In some yet further embodiments, some compositions of the presentinvention comprise oil thickeners in order to improve the tactileproperties of emulsions. Preferred oil thickeners include, but are notlimited to other solids (e.g., hydrophobic silicon oxides of theAEROSIL® type, which are available from Degussa AG). Examples ofadvantageous AEROSIL® oxide grades include AEROSIL® OX50, AEROSIL® 130,AEROSIL® 150, AEROSIL® 200, AEROSIL® 300, AEROSIL® 380, AEROSIL® MOX 80,AEROSIL® MOX 170, AEROSIL® COK 84, AEROSIL® R 202, AEROSIL® R 805,AEROSIL® R 812, AEROSIL® R 972, AEROSIL® R 974 and AEROSIL® R976.

In some additional embodiments, some personal care compositions of thepresent invention comprise at least one “metal soap” (i.e., a salt of ahigher fatty acid, with the exception of alkali metal salt), which arefunction as oil thickeners. Examples of such metal soaps include, butare not limited to aluminum stearate, zinc stearate and/or magnesiumstearate.

A variety of anionic surfactants are also useful herein (See e.g., U.S.Pat. No. 3,929,678). Exemplary anionic surfactants include, but are notlimited to alkoyl isethionates (e.g., C₁₂-C₃₀), alkyl and alkyl ethersulfates and salts thereof, alkyl and alkyl ether phosphates and saltsthereof, alkyl methyl taurates (e.g., C₁₂-C₃₀), and soaps (e.g.,substituted alkylamine and alkali metal salts, e.g., sodium or potassiumsalts) of fatty acids.

Amphoteric and zwitterionic surfactants are also useful herein. Examplesof preferred amphoteric and zwitterionic surfactants which find use inthe compositions of the present invention are those which are broadlydescribed as derivatives of aliphatic secondary and tertiary amines inwhich the aliphatic radical can be straight or branched chain andwherein one of the aliphatic substituents contains from about 8 to about22 carbon atoms (preferably C₈-C₁₈) and one contains an anionic watersolubilizing group (e.g., carboxy, sulfonate, sulfate, phosphate, orphosphonate). Examples, include but are not limited to alkyl iminoacetates and iminodialkanoates and aminoalkanoates, imidazolinium andammonium derivatives. Other suitable amphoteric and zwitterionicsurfactants are those selected from the group consisting of betaines,sultaines, hydroxysultaines, and branched and unbranched alkanoylsarcosinates, and mixtures thereof.

In some further embodiments, some personal care compositions comprise atleast one amphoteric and/or zwitterionic surfactant (e.g.,cocamidopropylbetain) and/or moisturizer (e.g. betain). Examples ofamphoteric surfactants that find use in such embodiments of the presentinvention include but are not limited to acyl/dialkylethylenediamine(e.g., sodium acylamphoacetate), disodium acylamphodipropionate,disodium alkylamphodiacetate, sodium acylamphohydroxypropylsulfonate,disodium acylamphodiacetate, sodium acylamphopropionate, N-alkylaminoacids, for example aminopropylalkylglutamide, alkylaminopropionic acid,sodium alkylimidodipropionate, and lauroamphocarboxyglycinate.

In some embodiments, the amount of surface- or interface-activesubstances (one or more compounds) in the preparations is preferablybetween about 0.001 and about 30% by weight, and more preferably betweenabout 0.05 and about 20% by weight, in most preferably between about 1and about 10% by weight, based on the total weight of the preparation.

In some yet additional embodiments, the active ingredients (one or morecompounds) comprise at least one lipophilic active ingredient. In someembodiments, these lipophilic active ingredients are selected from thegroup consisting of acetylsalicylic acid, atropine, azulene,hydrocortisone and derivatives thereof (e.g.,hydrocortisone-17-valerate), B vitamins, D vitamins, vitamin B₁, vitaminB₁₂, vitamin D₁, retinoid, bisabolol, unsaturated fatty acids (e.g., theessential fatty acids often also referred to as “vitamin F”),γ-linolenic acid, oleic acid, eicosapentenoic acid, docosahexenoic acidand derivatives thereof, chloramphenicol, caffeine, prostaglandins,thymol, camphor, extracts or other products of a vegetable and animalorigin (e.g. evening primrose oil, borrage oil or currant seed oil, fishoils, cod-liver oil), and ceramides and ceramide-like compounds, etc. Insome embodiments, the active ingredient(s) are refatting substances(e.g., purcellin oil, EUCERIT® and/or NEROCERIT®).

In further embodiments, some emulsions of the present invention includea silicone containing emulsifier or surfactant. A wide variety ofsilicone emulsifiers find use herein. These silicone emulsifiers aretypically organically modified organopolysiloxanes, also known to thoseskilled in the art as silicone surfactants. Useful silicone emulsifiersinclude, but are not limited to dimethicone copolyols. These materialsare polydimethyl siloxanes which have been modified to include polyetherside chains such as polyethylene oxide chains, polypropylene oxidechains, mixtures of these chains and polyether chains containingmoieties derived from both ethylene oxide and propylene oxide. Otherexamples include alkyl-modified dimethicone copolyols (i.e., compoundswhich contain C₂-C₃₀ pendant side chains). Still other usefuldimethicone copolyols include materials having various cationic,anionic, amphoteric, and zwitterionic pendant moieties.

In some embodiments, the compositions of the present invention compriseat least one polymeric thickening agent. The polymeric thickening agentsuseful herein preferably have a number average molecular weight ofgreater than about 20,000, more preferably greater than about 50,000,and most preferably greater than about 100,000. In some embodiments, thecompositions of the present invention comprise from about 0.01% to about10%, preferably from about 0.1% to about 8% and more preferably fromabout 0.2% to about 5% by weight of the composition of the polymericthickening agent or mixtures thereof.

Preferred polymer thickening agents for use herein include, but are notlimited to non-ionic thickening agents and anionic thickening agents ormixtures thereof. Suitable non-ionic thickening agents include, but arenot limited to polyacrylamide polymers, crosslinkedpoly(N-vinylpyrrolidones), polysaccharides, natural or synthetic gums,polyvinylpyrrolidone and polyvinylalcohol. Suitable anionic thickeningagents include, but are not limited to acrylic acid/ethyl acrylatecopolymers, carboxyvinyl polymers and crosslinked copolymers of alkylvinyl ethers and maleic anhydride. Commercially available thickeners(e.g., Carbopol; Noveon) find use in some embodiments of the presentinvention. Suitable Carbopol resins may be hydrophobically modified, andother suitable resins are described in WO98/22085, or mixtures thereof.

In some embodiments of the present invention, the water phase has a gelcharacter which, in addition to an effective content of compounds andsolvents (as appropriate) preferably comprises water, further organicand/or inorganic thickeners, and/or hydrocolloids.

In some embodiments, inorganic thickeners are selected from the groupconsisting of modified, unmodified, naturally occurring, and syntheticphyllosilicates. Although it is generally preferable to use purecomponents, in some embodiments, mixtures of different modified and/orunmodified phyllosilicates find use in various compositions of thepresent invention. As generally known in the art, phyllosilicates aresilicates and alumosilicates in which the silicate or aluminate unitsare linked together via three Si—O— or Al—O— bonds and form a wavy sheetor layer structure. The fourth Si—O— or Al—O— valence is saturated bycations. Relatively weak electrostatic interactions (e.g. hydrogenbridge bonds), exist between the individual layers. The layer structureis largely defined by strong, covalent bonds. The stochiometry of thesheet silicates is (Si₂O₅ ²⁻) for pure silicate structures and(Al_(m)Si²⁻ _(m)O₅ ^((2+m)−)) for alumosilicates, wherein “m” is anumber greater than zero and less than 2. In some embodiments in whichalumosilicates are present in the absence of pure silicates, each Si⁴⁺group replaced by Al³⁺ requires another singly charged cation toneutralize the charge. The charge balance is preferably evened out byH⁺, alkali metal ions or alkaline earth metal ions. In alternativeembodiments, aluminum is used as a counterion. In contrast to thealumosilicates, these compounds are referred to as “aluminum silicates.”“Aluminum alumosilicates,” in which aluminum is present both in thesilicate network, and also as counterion, also find use in someembodiments of the present invention.

Phyllosilicates are well known in the art (See e.g., Hollemann et al.,Lehrbuch der Anorganischen Chemie [Textbook of Inorganic Chemistry], 91st-100th Ed., Walter de Gruyter—Verlag [1985]; Remy, Lehrbuch derAnorganischen Chemie, 12^(th) Ed., Akademische Verlagsgesellschaft,Leipzig [1965]). The layer structure of montmorillonite is also known(See, Römpps Chemie-Lexikon, Franckh'sche Verlagshandlung, W. Keller &Co., Stuttgart, 8^(th) Ed., [1985], p. 2668 f). Examples ofphyllosilicates include the following (montmorillonite is the mainmineral comprising the naturally-occurring bentonites);

Montmorillonite Na_(0.33)((Al_(1.67)Mg_(0.33))(OH)₂(S_(i4)O₁₀)) oftensimplified: Al₂O₃*4SiO₂*H₂O*nH₂O or Al₂[(OH)₂/Si₄O₁₀]•n H₂O KaoliniteAl₂(OH)₄(Si₂O₅) Illite (K,H₃O)_(y)(Mg₃(OH)₂(Si_(4-y)Al_(y)O₁₀)) or(K,H₃O)_(y)(Al₂(OH)₂(Si_(4-y)Al_(y)O₁₀)) where y = 0.7-0.9 Beidellite(Ca,Na)_(0.3)(Al₂(OH)₂(Al_(0.5)Si_(3.5)O₁₀)) NontroniteNa_(0.33)(Fe₂(OH)₂(Al_(0.33)S_(i3.67)O₁₀)) Saponite(Ca,Na)_(0.33)((Mg,Fe)₃(OH)₂(Al_(0.33)Si_(3.67)O₁₀)) HectoriteNa_(0.33)((Mg,Li)₃(OH,F)₂(Si₄O₁₀))

In some preferred embodiments, inorganic gel formers including but notlimited to aluminum silicates, such as the montmorillonites (bentonites,hectorites and derivatives thereof, such as quaternium-18 bentonite,quaternium-18 hectorites, stearalkonium bentonites and stearalkoniumhectorites), and also magnesium-aluminum silicates (VEEGUM® grades), andsodium-magnesium silicates (LAPONITE® grades) find use in the presentinvention.

Montmorillonites represent clay minerals which are a type ofdioctahedral smectites, and are masses which swell in water, but do notbecome plastic. The layer packets in the three-layer structure of themontmorillonites can swell as a result of reversible incorporation ofwater (in a 2- to 7-fold amount) and other substances, such as, forexample, alcohols, glycols, pyridine, picoline, ammonium compounds,hydroxy-aluminosilicate ions etc. The chemical formula given aboveprovides just an approximation of the formula, as montmorillonites havea large capacity for ion exchange. For example, Al can be replaced byMg, Fe²⁺, Fe³⁺, Zn, Pb (e.g., from harmful substances in waste waters),Cr, Cu and other elements. The resulting negative charge of theoctahedral layers is compensated for by the presence of cations, inparticular Na⁺ (i.e., sodium montmorillonite) and Ca²⁺ (i.e., calciummontmorillonite, a compound that is only swellable to a very smallextent) in interlayer positions.

In alternative embodiments, synthetic magnesium silicates and/orbentonites find use in the present invention, including but not limitedto such commercially available compounds as OPTIGEL® (Süd-Chemie). Asindicated above, in some embodiments, aluminum silicates such as thecommercially available VEEGUM® (R.T. Vanderbilt Comp., Inc), find use inthe present invention. Various VEEGUM® grades which find use in variousembodiments of the present invention are provided below.

VEEGUM ® Grades Regular Grade HV K HS S-728 SiO₂ 55.5 56.9 64.7 69.065.3 MgO 13.0 13.0 5.4 2.9 3.3 Al₂O₃ 8.9 10.3 14.8 14.7 17.0 Fe₂O₃ 1.00.8 1.5 1.8 0.7 CaO 2.0 2.0 1.1 1.3 1.3 Na₂O 2.1 2.8 2.2 2.2 3.8 K₂O 1.31.3 1.9 0.4 0.2 Ashing loss 11.1 12.6 7.6 5.5 7.5

The above products swell in water to form viscous gels, which have analkaline reaction. The organophilization of montmorillonite orbentonites (exchange of the interlayer cations for quaternaryalkylammonium ions) produces products (bentones) which are preferablyused for dispersion in organic solvents and oils, fats, ointments, inks,surface coatings and in detergents.

BENTONE® is a trade name for various neutral and chemically inertgelling agents which are constructed from long-chain, organic ammoniumsalts and specific types of montmorillonite. BENTONE® gelling agentsswell in organic media, which cause the media to also swell. The gelsare resistant to diluted acids and alkalis, although they partially losetheir gelling properties upon prolonged contact with strong acids andalkalis. Because of their organophilic character, BENTONE® gellingagents are only wettable by water with difficulty. There are variousBENTONE® gelling agent grades commercially available, including thosesold by Kronos Titan: BENTONE® 27, an organically modifiedmontmorillonite; BENTONE® 34 (dimethyldioctylammonium bentonite;prepared in accordance with U.S. Pat. No. 2,531,427, incorporated hereinby reference, which because of its lipophilic groups, swells morereadily in lipophilic medium than in water); BENTONE® 38, an organicallymodified montmorillonite, available as a cream-colored to white powder;BENTONE® LT, a purified clay mineral; BENTONE® Gel MIO, an organicallymodified montmorillonite which is supplied as a very fine suspension inmineral oil (SUS-71) (10% bentonite, 86.7% mineral oil and 3.3% wettingagent); BENTONE® Gel IPM, an organically modified bentonite which issuspended in isopropyl myristate (10% bentonite, 86.7%isopropylmyristate, 3.3% wetting agent); BENTONE® Gel CAO, anorganically modified montmorillonite which is taken up in castor oil(10% bentonite, 86.7% castor oil and 3.3% wetting agent); BENTONE® GelLantrol, an organically modified montmorillonite which, in paste form,is intended for the further processing, in particular for thepreparation, of cosmetic compositions; 10% bentonite, 64.9 LANTROL®(wool wax oil), 22.0 isopropyl myristate, 3.0 wetting agent and 0.1propyl p-hydroxybenzoate; BENTONE® Gel Lan I, a 10% strength BENTONE® 27paste in a mixture of wool wax USP and isopropyl palmitate; BENTONE® GelLan II, a bentonite paste in pure liquid wool wax; BENTONE® Gel NV, a15% strength BENTONE® 27 paste in dibutyl phthalate; BENTONE® Gel OMS, abentonite paste in Shellsol T.; BENTONE® Gel OMS 25, a bentonite pastein isoparaffinic hydrocarbons (IDOPAR® H); and BENTONE® Gel IPP, abentonite paste in isopropyl palmitate.

“Hydrocolloid” is the technological abbreviation for the more correctname “hydrophilic colloid.” Hydrocolloids are macromolecules which havea largely linear structure and intermolecular forces of interactionwhich permit secondary and primary valence bonds between the individualmolecules to form a recticular structure. Some hydrocolloids arewater-soluble natural or synthetic polymers which, in aqueous systems,form gels or viscous solutions. These compounds increase the viscosityof water by either binding water molecules (hydration), or by absorbingand encapsulating the water into their interwoven macromolecules, whilerestricting the mobility of water. These water-soluble polymersrepresent a large group of natural and synthetic polymers that arechemically very different, but which share a common feature in theirsolubility in water or aqueous media. A prerequisite for this is thatthese polymers have a number of hydrophilic groups sufficient forsolubility in water and are not too greatly crosslinked. Thesehydrophilic groups can be nonionic, anionic or cationic in nature, forexample as follows:

In some preferred embodiments, the group of the cosmetically anddermatologically relevant hydrocolloids are divided into the followinggroups: organic, natural compounds (e.g., agar agar, carrageen,tragacanth, gum arabic, alginates, pectins, polyoses, guar flour, carobbean flour, starch, dextrins, gelatins, and casein); organic, modifiednatural substances (e.g., carboxymethylcellulose and other celluloseethers, hydroxyethylcellulose and hydroxypropylcellulose andmicrocristalline cellulose); organic, completely synthetic compounds(e.g., polyacrylic and polymethacrylic compounds, vinyl polymers,polycarboxylic acids, polyethers, polyimines, polyamides, andpolyurethanes); and inorganic compounds (e.g., polysilicic acids, clayminerals, such as montmorillonites, zeolites, and silicas).

In alternative embodiments, ethylcelluloses find use in compositions ofthe present invention as stabilizers. Ethylcelluloses are characterizedby the following structure. In this structure, the Rs are either ethylgroups or hydrogen atoms.

In some preferred embodiments, the degree of ethylation in theethylcellulose is from about 2.0 to about 3.0, corresponding to about 40to about 55%, and more preferably about 48.0 to about 49.5% ethylation.The average molecular mass is preferably chosen such that the viscosityof an approximately 5% strength solution in a mixture of 80 parts oftoluene and 20 parts of ethanol at 25° C. is 3 to 110 mPas, and morepreferably 9 to 11 mPas. In some particularly preferred embodiments, theaverage molar mass is from about 100,000 to about 400,000 g/mol. In somepreferred embodiments, the ethylcellulose concentration in compositionsof the present invention ranges from about 0.1 to about 10% by weight,based on the total weight of the preparations. Various ethylcellulosesfind use in the present invention, including but not limited to thosethat are commercially available (e.g., ETHOCEL® Standard 10 Premium; DowChemicals).

In yet additional embodiments, microcristalline cellulose finds use ashydrocolloid in compositions of the present invention. Variousmicrocrystalline cellulose preparations find use in the presentinvention, including but not limited to those that are commerciallyavailable (e.g., AVICEL®, such as AVICEL® RC-591, as well as AVICEL®RC/CL; AVICEL® CE-15; and AVICEL® 500; FMC Corporation Food andPharmaceutical Products). In some particularly preferred embodiments,AVICEL® RC-591 (a modified microcristalline cellulose which is made upof 89% microcrystalline cellulose and 11% sodium carboxymethylcellulose)finds use in the present invention.

Additional hydrocolloids that find use in the present invention includemethylcelluloses (i.e., methylesters of cellulose). These compounds arecharacterized by the following structural formula

in which R can be a hydrogen or a methyl group.

Cellulose mixed ethers (generally referred to as methylcelluloses, whichcontain, in addition to a predominating content of methyl groups, also2-hydroxyethyl, 2-hydroxypropyl or 2-hydroxybutyl groups) also find usein some embodiments of the present invention. In some preferredembodiments, hydroxypropyl)methyl-celluloses (e.g., METHOCEL® E4M; DowChemical Co.) find use in the present invention.

In yet further embodiments sodium carboxymethylcellulose (i.e., thesodium salt of the glycolic ether of cellulose, for which R in the abovestructural formula may be hydrogen and/or CH₂—COONa) finds use in thepresent invention. In some preferred embodiments, sodiumcarboxymethylcellulose, also sometimes referred to as “cellulose gum”(e.g., NATROSOL® Plus 330 CS; Aqualon) finds use in the presentinvention.

In additional embodiments, xanthan (CAS No. 11138-66-2), (i.e., xanthangum), an anionic heteropolysaccharide generally formed by fermentationfrom maize sugar and isolated as potassium salt finds use in the presentinvention. It is produced by Xanthomonas campestris and some otherspecies under aerobic conditions and has a molecular weight of from2×10⁶ to 24×10⁶. Xanthan is formed from a chain having cellulose withside chains. The structure of the subgroups consists of glucose,mannose, glucuronic acid, acetate and pyruvate. The number of pyruvateunits determines the viscosity of the xanthan.

In still further embodiments, carrageen is used as a gel former incompositions of the present invention. This compound is an extract fromNorth Atlantic red algae (Florideae; Chondrus crispus and Gigartinastellata) that has a structure similar to that of agar. The term“carrageen” is frequently used in reference to a dried algae product and“carrageenan” is used in reference to the extract thereof. The carrageenprecipitated from the hot-water extract of the algae is a colorless tosand-colored powder with a molecular weight range from about 100,000 toabout 800,000 and a sulfate content of about 25%. Carrageen, which isvery readily soluble in warm water, forms a thixotropic gel uponcooling, even if the water content is 95-98%. The rigidity of the gel iseffected by the double helix structure of the carrageen.

In the case of carrageenan, three principle constituents aredifferentiated. The gel-forming “κ fraction” consists of D-galactose4-sulfate and 3,6-anhydro-α-D-galactose, which has alternate glycosidebonds in the 1,3- and 1,4 position (in contrast, agar contains3,6-anhydro-α-L-galactose). The nongelling “λ fraction” is composed of1,3-glycosidically linked D-galactose 2-sulfate and 1,4-bondedD-galactose-2,6-disulfate radicals, and is readily soluble in coldwater. Finally, “ι-carrageenan,” composed of D-galactose 4-sulfate in1,3 bond and 3,6-anhydro-α-D-galactose 2-sulfate in 1,4 bond, is bothwater-soluble and also gel-forming. The nature of any cations which arepresent (K⁺, NH₄ ⁺, Na⁺, Mg²⁺, Ca²⁺) also influences the solubility ofthe carrageens.

In yet additional embodiments, chitosan (i.e., partially deacylatedchitin) finds use in various compositions of the present invention.Chitosan has film-forming properties and is characterized as having asilky feel on the skin. One disadvantage for some uses, is its severestickiness on the skin which occurs in temporarily (usually) duringapplication. Due to this stickiness, some preparations are notacceptable to consumers. However, chitosan finds use in somepreparations, including hair care compositions, as it is better thanchitin in thickening and/or stabilizing, as well as improving theadhesion and water resistance of polymeric films. The use of chitosan iswell-known to those of skill in the personal care art (See e.g.,Fiedler, Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und angrenzendeGebiete, [Lexikon of auxiliaries for pharmacy, cosmetics and relatedfields], 3^(rd) edition, Editio Cantor, Aulendorf, [1989], p. 293).Chitosan is characterized by the following structural formula:

where n assumes values up to about 10 000, and X is either the acetylradical or hydrogen. Chitosan forms by deacetylation and partialdepolymerization (hydrolysis) of chitin, which is characterized by thestructural formula

Chitin is an essential constituent of the arthropod (e.g. insects,crabs, and spiders) ectoskeleton, and is also found in the connectiveand/or supporting tissues of other organisms (e.g. mollusks, algae, andfungi). In the region of about pH <6, chitosan is positively charged andin that range is also soluble in aqueous systems. It is incompatiblewith anionic raw materials. For this reason, in order to preparechitosan-containing oil-in-water emulsions, the use of nonionicemulsifiers is appropriate (See e.g., EP 776 657). In some preferredembodiments, the compositions of the present invention contain at leastone chitosans with a degree of deacetylation of at least about >25%, andmore preferably, a range of more than about 55 to about 99% (asdetermined by means of ¹H-NMR). In some embodiments, chitosans ofmolecular weights between about 10,000 and about 1,000,000, inparticular those with molecular weights between 100,000 and 1,000,000(determined by means of gel permeation chromatography) find use in thepresent invention.

In yet further embodiments, polyacrylates find use as gelling agents insome compositions of the present invention. Suitable polyacrylatesinclude but are not limited to acrylate-alkyl acrylate copolymers, inparticular those chosen from the group of carbomers or CARBOPOL®copolymers (B. F. Goodrich Co.). In particular, the acrylate-alkylacrylate copolymers that find use in some embodiments of the presentinvention have the following structure:

where R′ is a long-chain alkyl radical, and x and y represent numberswhich symbolize the respective stoichiometric proportion of each of thecomonomers.

In some embodiments, acrylate copolymers and/or acrylate-alkyl acrylatecopolymers, include but are not limited to those that are commerciallyavailable (e.g., CARBOPOL® 1382, CARBOPOL® 981, and CARBOPOL® 5984; B.F. Goodrich Co., and in particular, polyacrylates from the group ofCARBOPOL grades 980, 981, 1382, 2984, 5984 and Carbomer 2001). Inadditional embodiments, copolymers of C₁₀₋₃₀-alkyl acrylates and one ormore monomers of acrylic acid, of methacrylic acid or esters thereofwhich are crosslinked with an allyl ether of sucrose or an allyl etherof pentaerythrito find use in some embodiments of the present invention.

Compounds which carry the INCI name “Acrylates/C₁₀₋₃₀ Alkyl AcrylateCrosspolymer” also find use in some embodiments of the presentinvention. In some embodiments, commercially available polymers (e.g.,PEMULEN® TR1 and PEMULEN® TR2; B. F. Goodrich Co.) find use in someembodiments of the present invention, although it is not intended thatthe present invention be limited to any specific acrylate-containingcomposition.

In yet additional embodiments, compounds which carry the INCI name“ammonium acryloyldimethyltaurates/vinylpyrrolidone copolymers” find usein the present invention. These ammonium acryloyldimethyltaurate/vinylpyrrolidone copolymers have the empirical formula[C₇H₁₆N₂SO₄]_(n)[C₆H₉NO]_(m), which corresponds to the followingstructure:

Preferred species of this compound are listed in Chemical Abstractsunder the Registry numbers 58374-69-9, 13162-05-5 and 88-12-0, and arecommercially available (e.g., ARISTOFLEX®; Clariant GmbH). However, itis not intended that the present invention be limited to any particularspecies. In yet additional embodiments of the present invention,copolymers/crosspolymers comprising acryloyldimethyl taurate (e.g.,SIMUGEL® EG and SIMUGEL® EG; Seppic S.A.) find use in some compositionsof the present invention.

Additional completely synthetic hydrocolloids that find use in thepresent invention include, but are not limited to anionic polyurethaneswhich are soluble or dispersible in water and which are advantageouslyobtainable from:

Aa) at least one compound which contains two or more active hydrogenatoms per molecule,

Ab) at least one diol containing acid or salt groups, and

Ac) at least one diisocyanate.

In some preferred embodiments, the component Aa) is, in particular, adiol, aminoalcohol, diamine, polyesterol, polyetherol with anumber-average molecular weight of in each case up to 3000, or mixturesthereof, where up to 3 mol % of said compounds may be replaced by triolsor triamines. Preference is given to diols and polyesterdiols. Inparticular, the component Aa) comprises at least 50% by weight, based onthe total weight of the component Aa), of a polyesterdiol. Suitablepolyesterdiols are all those which are customarily used for thepreparation of polyurethanes, in particular the reaction products ofphthalic acid and diethylene glycol, isophthalic acid and1,4-butanediol, isophthalic acid/adipic acid and 1,6-hexanediol, andadipic acid and ethylene glycol or 5-NaSO₃-isophthalic acid, phthalicacid, adipic acid and 1,6-hexanediol.

Examples of diols which find use in some embodiments of the presentinvention include, but are not limited to ethylene glycol, propyleneglycol, butylene glycol, neopentyl glycol, polyetherols (e.g.,polyethylene glycols having molecular weights up to 3000), blockcopolymers of ethylene oxide and propylene oxide with number-averagemolecular weights of up to 3000, and block copolymers of ethylene oxide,propylene oxide and butylene oxide which contain the copolymerizedalkylene oxide units in randomly distributed manner or in the form ofblocks. Preference is given to ethylene glycol, neopentyl glycol, di-,tri-, tetra-, penta- or hexaethylene glycol. Other diols which find useinclude poly(α-hydroxycarboxylic acid)diols.

Suitable aminoalcohols that find use in some embodiments of the presentinvention include but are not limited to 2-aminoethanol,2-(N-methylamino)ethanol, 3-aminopropanol, and 4-aminobutanol.

In some embodiments, diamines such as ethylenediamine, propylenediamine,1,4-diaminobutan, 1,6-diaminohexane, and α,ω-diamines which can beprepared by amination of polyalkylene oxides with ammonia find use insome compositions of the present invention.

Component Ab) is, in particular, dimethylolpropanoic acid or a compoundwith the formula:

where RR is in each case a C₂-C₁₈-alkylene group and Me is Na or K.

Component Ac) is, in particular, hexamethylene diisocyanate, isophoronediisocyanate, methyldiphenyl isocyanate (MDI), and/or tolylenediisocyanate.

In some embodiments, the polyurethanes are obtained by reacting thecompounds of groups Aa) and Ab) under an inert-gas atmosphere in aninert solvent at temperatures of from 70 to 130° C. with the compoundsof group Ac). This reaction can be carried out, where appropriate, inthe presence of chain extenders in order to prepare polyurethanes withrelatively high molecular weights. As is customary in the preparation ofpolyurethanes, the components [(Aa)+(Ab)]:Ac are advantageously used inthe molar ratio of from 0.8 to 1.1:1. The acid number of thepolyurethanes is determined by the composition and the concentration ofthe compounds of component (Ab) in the mixture of components (Aa) and(Ab).

In some embodiments, the polyurethanes have K values according to H.Fikentscher (determined in 0.1% strength by weight solutions inN-methylpyrrolidone at 25° C. and pH 7) of from about 15 to about 100,and preferably about 25 to about 50. The K value (i.e., “intrinsicviscosity”), is a parameter which is easy to determine by means ofviscosity measurements of polymer solutions and is therefore frequentlyused in the industrial sector for characterizing polymers. Polyurethanescontaining acid groups that find use in some embodiments of the presentinvention include, but are not limited to polyurethanes that arewater-soluble or dispersible without the aid of emulsifiers afterpartial or complete neutralization. The salts of the polyurethanesgenerally have better solubility or dispersibility in water than theunneutralized polyurethanes. Bases which find use for the neutralizationof the polyurethanes include alkali metal bases (e.g., sodium hydroxidesolution, potassium hydroxide solution, soda, sodium hydrogencarbonate,potassium carbonate or potassium hydrogen carbonate) and alkaline earthmetal bases (e.g., calcium hydroxide, calcium oxide, magnesium hydroxideor magnesium carbonate, and ammonia and amines). In some embodiments,2-amino-2-methylpropanol, diethylaminopropylamine and triisoproanolaminefind particular use in the neutralization of the polyurethanescontaining acid groups. In yet additional embodiments, theneutralization of the polyurethanes containing acid groups is carriedout using mixtures of two or more bases (e.g. mixtures of sodiumhydroxide solution and triisopropanolamine). Depending on the intendeduse, neutralization is partial (e.g. about 20 to about 40%) or complete(i.e., 100%). These polymers and their preparation are described in moredetail in DE-A-42 25 045, incorporated herein by reference.

B. Water-soluble or -dispersible cationic polyurethanes and polyureasof:

Ba) at least one diisocyanate, which may have already been reactedbeforehand with one or more compounds which contain two or more activehydrogen atoms per molecule, and

Bb) at least one diol, primary or secondary amino alcohol, primary orsecondary diamine or primary or secondary triamine with one or moretertiary, quaternary or protonated tertiary amino nitrogen atoms.

Preferred diisocyanates are as given above under A). Compounds with twoor more active hydrogen atoms are diols, aminoalcohols, diamines,polyesterols, polyamidediamines and polyetherols. Suitable compounds ofthis type are as given above under A).

The polyurethanes are prepared as described above under A). Chargedcationic groups can be produced in the polyureas from the tertiary aminonitrogen atoms present either by protonation, (e.g., with carboxylicacids, such as lactic acid), or by quaternization (e.g. with alkylatingagents, such as C₁ to C₄-alkyl halides) or sulfates. Examples of suchalkylating agents include, but are not limited to ethyl chloride, ethylbromide, methyl chloride, methyl bromide, dimethyl sulfate and diethylsulfate. These polymers and their preparation are described in moredetail in DE-A-42 41 118, which is incorporated herein by reference.

C. Linear polyurethanes with carboxylate groups of:

Ca) a 2,2-hydroxymethyl-substituted carboxylic acid of the formula

 in which RR′ is a hydrogen atom or a C₁-C₂₀-alkyl group, which is usedin an amount which suffices for about 0.35 to about 2.25milliequivalents of carboxyl groups to be present in the polyurethaneper g of polyurethane,

Cb) about 10 to about 90% by weight, based on the weight of thepolyurethane, of one or more organic compounds with not more than twoactive hydrogen atoms and

Cc) one or more organic diisocyanates.

In some preferred embodiments, the carboxyl groups present in thepolyurethane are, finally, at least partially neutralized with asuitable base. These polymers and their preparation are described inEP-A-619 111, incorporated herein by reference.

D. Carboxyl-containing polycondensation products of anhydrides of tri-or tetracarboxylic acids and diols, diamines or aminoalcohols(polyesters, polyamides or polyester amides). These polymers and theirpreparation are described in more detail in DE-A-42 24 761, incorporatedherein by reference.

E. Polyacrylates and polymethacrylates, as are described in more detailin DE-A-43 14 305, 36 27 970 and 29 17 504, all of which areincorporated herein by reference.

The polymers used in some embodiments of the present invention have a Kvalue of from about 15 to about 100, and more preferably from about 25to about 50. The polymers are generally present in the composition in anamount in the range from about 0.2 to about 20% by weight, based on thetotal weight of the compositions. The salt is used in an amounteffective for improving the exchangeability of the polymers. The salt isgenerally used in an amount of from about 0.02 to about 10% by weight,and more preferably from about 0.05 to about 5% by weight, and inparticular, from about 0.1 to about 3% by weight, based on the totalweight of the composition.

The total amount of one or more hydrocolloids in some embodiments of thepersonal care compositions of the present invention is less than about5% by weight, preferably between about 0.05 and about 3.0% by weight,and more preferably between about 0.1 and about 1.0% by weight, based onthe total weight of the preparations.

In some embodiments, the present compositions comprise at least onesilicone oil phase. Silicone oil phase(s) generally comprises from about0.1% to about 20%, preferably from about 0.5% to about 10%, and morepreferably from about 0.5% to about 5%, of the composition. The siliconeoil phase preferably comprises one or more silicone components.

In some embodiments, silicone components are fluids, including straightchain, branched and cyclic silicones. Suitable silicone fluids usefulherein include silicones inclusive of polyalkyl siloxane fluids,polyaryl siloxane fluids, cyclic and linear polyalkylsiloxanes,polyalkoxylated silicones, amino and quaternary ammonium modifiedsilicones, polyalkylaryl siloxanes or a polyether siloxane copolymer andmixtures thereof. Volatile, as well as non-volatile silicone fluids finduse herein. Silicone fluids generally have an average molecular weightof less than about 200,000. In preferred embodiments, suitable siliconefluids have a molecular weight of about 100,000 or less, preferablyabout 50,000 or less, and more preferably about 10,000 or less.Preferably the silicone fluid is selected from silicone fluids having aweight average molecular weight in the range from about 100 to about50,000 and preferably from about 200 to about 40,000. Typically,silicone fluids have a viscosity ranging from about 0.65 to about600,000 mm²s⁻¹, preferably from about 0.65 to about 10,000 mm²·s⁻¹ at25° C. The viscosity can be measured by means of a glass capillaryviscometer as set forth in Dow Corning Corporate Test Method CTM0004,Jul. 29, 1970. Suitable polydimethyl siloxanes that can be used hereininclude commercially available compounds (e.g., from the GeneralElectric Company and Dow Corning). Also useful are essentiallynon-volatile polyalkylarylsiloxanes, for example,polymethylphenylsiloxanes, having viscosities of about 0.65 to 30,000mm²s⁻¹ at 25° C. (General Electric Company or from Dow Corning). Cyclicpolydimethylsiloxanes suitable for use herein are those having a ringstructure incorporating from about 3 to about 7 (CH₃)₂SiO moieties,preferably about 5 or more.

In additional embodiments, silicone gums find use herein. In somepreferred embodiments, a silicone oil phase comprises a silicone gum ora mixture of silicones including the silicone gum. Typically, siliconegums have a viscosity at 25° C. in excess of about 1,000,000 mm²s⁻¹. Thesilicone gums include dimethicones as known in the art (See e.g., U.S.Pat. No. 4,152,416; and Noll, Chemistry and Technology of Silicones,Academic Press, New York [1968]). Silicone gums such as those describedin General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE54 and SE 76, also find use in the present invention. Specific examplesof silicone gums include polydimethylsiloxane, (polydimethylsiloxane)(methylvinylsiloxane) copolymer,poly(dimethylsiloxane)(diphenyl)(methylvinylsiloxane) copolymer andmixtures thereof. Preferred silicone gums for use herein are siliconegums having a molecular weight of from about 200,000 to about 4,000,000selected from dimethiconol, dimethicone copolyol, dimethicone andmixtures thereof.

In some embodiments, a silicone phase herein preferably comprises asilicone gum incorporated into the composition as part of a siliconegum-fluid blend. When the silicone gum is incorporated as part of asilicone gum-fluid blend, the silicone gum preferably constitutes fromabout 5% to about 40%, especially from about 10% to 20% by weight of thesilicone gum-fluid blend. Suitable silicone gum-fluid blends herein aremixtures consisting essentially of:

(i) a silicone having a molecular weight of from about 200,000 to about4,000,000 selected from dimethiconol, fluorosilicone and dimethicone andmixtures thereof; and

(ii) a carrier which is a silicone fluid, the carrier having a viscosityfrom about 0.65 mm²s⁻¹ to about 100 mm²s⁻¹,

wherein the ratio of i) to ii) is from about 10:90 to about 20:80 andwherein said silicone gum-based component has a final viscosity of fromabout 100 mm²s⁻¹ to about 100,000 mm²s⁻¹, preferably from 500 mm²s⁻¹ toabout 10,000 mm²s⁻¹.

Further silicone components suitable for use in a silicone oil phaseherein include crosslinked polyorganosiloxane polymers, optionallydispersed in a fluid carrier. In general, when present the crosslinkedpolyorganosiloxane polymers, together with its carrier (if present)comprises from about 0.1% to about 20%, preferably from about 0.5% toabout 10%, and more preferably from about 0.5% to about 5% of thecomposition. Such polymers comprise polyorganosiloxane polymerscrosslinked by a crosslinking agent (See e.g., WO98/22085). Examples ofsuitable polyorganosiloxane polymers for use herein include, but are notlimited to methyl vinyl dimethicone, methyl vinyl diphenyl dimethiconeand methyl vinyl phenyl methyl diphenyl dimethicone.

Another class of silicone components suitable for use in a silicone oilphase herein includes polydiorganosiloxane-polyoxyalkylene copolymerscontaining at least one polydiorganosiloxane segment and at least onepolyoxyalkylene segment (See e.g., WO98/22085). Suitablepolydiorganosiloxane-polyalkylene copolymers are available commerciallyunder the tradenames BELSIL® from Wacker-Chemie GmbH. A particularlypreferred copolymer fluid blend for use herein includes Dow CorningDC3225C which has the CTFA designation Dimethicone/Dimethicone copolyol.

In further embodiments, compositions of the present invention comprisean organic sunscreen. In some embodiments, suitable sunscreens have UVAabsorbing properties, while others have UVB absorbing properties, andstill others comprise a mixture thereof. The exact amount of thesunscreen active varies, depending upon the desired Sun ProtectionFactor (i.e., the “SPF”) of the composition, as well as the desiredlevel of UV protection. SPF is a commonly used measure ofphotoprotection of a sunscreen against erythema. The SPF is defined as aratio of the ultraviolet energy required to produce minimal erythema onprotected skin to that required to produce the same minimal erythema onunprotected skin in the same individual. Amounts of the sunscreen usedare preferably from about 2% to about 20%, and more preferably fromabout 4% to about 14%. Suitable sunscreens include, but are not limitedto those approved for use in the United States, Japan, Europe andAustralia. The compositions of the present invention preferably comprisean SPF of about 2 to about 30, preferably about 4 about 30, and morepreferably about 4 to about 15.

In some embodiments, the compositions of the present invention compriseone or more UVA absorbing sunscreen actives that absorb UV radiationhaving a wavelength of from about 320 nm to about 400 nm. Suitable UVAabsorbing sunscreen actives include, but are not limited todibenzoylmethane (See e.g., Lowe and Shaath (eds.), Sunscreens:Development, Evaluation, and Regulatory Aspects, Marcel Dekker, Inc.)derivatives, anthranilate derivatives such as methylanthranilate andhomomethyl, 1-N-acetylanthranilate, and mixtures thereof. The UVAabsorbing sunscreen active is preferably present in an amount sufficientto provide broad spectrum UVA protection either independently, or incombination with, other UV protective actives which may be present inthe composition.

Suitable UVA sunscreen actives include dibenzoylmethane sunscreenactives and their derivatives. They include, but are not limited to,those selected from 2-methyldibenzoylmethane, 4-methyldibenzoylmethane,4-isopropyldibenzoylmethane, 4-tert-butyldibenzoylmethane,2,4-dimethyldibenzoylmethane, 2,5-dimethyldibenzoylmethane,4,4′-diisopropylbenzoylmethane,4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane,2-methyl-5-isopropyl-4′-methoxydibenzoylmethane,2-methyl-5-tert-butyl-4′-methoxy-dibenzoylmethane,2,4-dimethyl-4′-methoxydibenzoylmethane,2,6-dimethyl-4′-tert-butyl-4′methoxydibenzoylmethane, and mixturesthereof. Preferred dibenzoyl sunscreen actives include those selectedfrom 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane,4-isopropyldibenzoylmethane, and mixtures thereof. A preferred sunscreenactive is 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane.

The sunscreen active 4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane,which is also known as butyl methoxydibenzoylmethane or “avobenzone,” iscommercially available under the names of Parsol® 1789 from GivaudanRoure (International) S. A., and Eusolex® 9020 from Merck & Co., Inc.The sunscreen 4-isoproplydibenzoylmethane, which is also known asisopropyldibenzoylmethane, is commercially available from Merck underthe name of Eusolex® 8020.

In some embodiments, the compositions of the present invention furtherinclude one or more UVB sunscreen actives that absorb(s) UV radiationhaving a wavelength of about 290 nm to about 320 nm. The compositionscomprise an amount of the UVB sunscreen active that is safe andeffective in providing UVB protection either independently, or incombination with, other UV protective actives which may be present inthe compositions. The compositions comprise from about 0.1% to about20%, preferably from about 0.1% to about 12%, and more preferably fromabout 0.5% to about 8% by weight, of each UVB absorbing organicsunscreen, or as mandated by the relevant regulatory authority(s).

A variety of UVB sunscreen actives are suitable for use herein (Seee.g., U.S. Pat. Nos. 5,087,372; 5,073,371; 5,073,372; 4,937,370; and4,999,186). Preferred UVB sunscreen actives are selected from2-ethylhexyl-2-cyano-3,2-ethylhexyl N,N-dimethyl-p-aminobenzoate,p-amino-benzoic acid, oxybenzone, homomethyl salicylate, octylsalicylate, 4,4′-methoxy-t-butyldibenzoylmethane, 4-isopropyldibenzoylmethane, 3-benzylidene camphor, 3-(4-methylbenzylidene)camphor, 3-diphenylacrylate, 2-phenyl-benzimidazole-5-sulphonic acid(PBSA), cinnamate esters and their derivatives such as2-ethylhexyl-p-methoxycinnamate, salicylate esters and their derivativessuch as triethanolamine salicylate, ethylhexyl salicylate, octyldimethylpara-aminobenzoic acid, camphor derivatives and their derivatives, andmixtures thereof. Preferred organic sunscreen actives include2-ethylhexyl-2-cyano-3,3-diphenylacrylate,2-phenyl-benzimidazole-5-sulphonic acid (PBSA),octyl-p-methoxycinnamate, and mixtures thereof. Salt and acidneutralized forms of the acidic sunscreens are also useful herein.

Thus, in some embodiments, the present invention provides compositionscomprising any organic UV-A and UV-B filter, for example but not limitedto the following:

CAS-Nr. Nr. Compound (=Acid) 1 4-Aminobenzoicacid 150-13-0 23-(4′-Trimethylammonium)-benzylidenbornan-2-on- 52793-97-2 methylsulfate3 3,3,5-Trimethyl-cyclohexyl-salicylate 118-56-9 (Homosalatum) 42-Hydroxy-4-methoxy-benzophenon 131-57-7 (Oxybenzonum) 52-Phenylbenzimidazol-5-sulfonic acid and their Calcium-, 27503-81-7Sodium- and Triethanolaminosalts 63,3′-(1,4-Phenylendimethin)-bis(7,7-dimethyl- 90457-82-22-oxobicyclo[2.2.1]heptan-1-methansolfonicacid) and salts thereof 74-Bis(polyethoxy)amino-benzoesaurepolyethoxy-ethylester 113010-52-9 84-Dimethylamino-benzoicacid-2-ethylhexylester 21245-02-3 9Salicylicacid-2-ethylhexylester 118-60-5 104-Methoxy-cinnamicacid-2-isoamylester 71617-10-2 114-Methoxy-cinnamicacid-2-ethylhexylester 5466-77-3 122-Hydroxy-4-methoxy-benzophenon-5-sulfonicacid- 4065-45-6(Sulisobenzonum) and the sodiumsalt 133-(4′-Sulfobenzyliden)-bornan-2-on and salts thereof 58030-58-6 143-Benzylidenbornan-2-on 16087-24-8 151-(4′-Isopropylphenyl)-3-phenylpropan-1,3-dion 63260-25-9 164-Isopropylbenzylsalicylat 94134-93-7 17 3-Imidazol-4-yl-acrylicacid undihr Ethylester 104-98-3 18 2-Cyano-3,3-diphenylacrylicacidethylester5232-99-5 19 2-Cyano-3,3-diphenylacrylicacid-2′-ethylhexylester6197-30-4 20 Menthyl-o-aminobenzoat oder: 134-09-85-Methyl-2-(1-methylethyl)-2-aminobenzoat 21 Glyceryl p-aminobenzoatoder: 136-44-7 4-Aminobenzoicacid-1-glyceryl-ester 222,2′-Dihydroxy-4-methoxybenzophenon (Dioxybenzone) 131-53-3 232-Hydroxy-4-methoxy-4-methylbenzophenon 1641-17-4 (Mexenon) 24Triethanolamin Salicylat 2174-16-5 25 Dimethoxyphenylglyoxalsäure oder:4732-70-1 3,4-dimethoxy-phenyl-glyoxal-saures Natrium 263-(4′Sulfobenzyliden)-bornan-2-on und seine Salze 56039-58-8 274-tert.-Butyl-4′-methoxy-dibenzoylmethan 70356-09-1 282,2′,4,4′-Tetrahydroxybenzophenon 131-55-5 292,2′-Methylen-bis-[6-(2H-benzotriazol-2-yl)-4-(1,1,3,3,- 103597-45-1tetramethylbutyl)phenol] 30 2,2′-(1,4-Phenylen)-bis-1H-benzimidazol-4,6-180898-37-7 disulfonicacid, Sodiumsalt 312,4-bis-[4-(2-Ethylhexyloxy)-2-hydroxy]phenyl- 187393-00-66-(4-methoxyphenyl)-(1,3,5)-triazin 32 3-(4-Methylbenzyliden)-campher36861-47-9 33 4-Bis(polyethoxy)paraaminobenzoicacidpolyethoxyethylester113010-52-9 34 2,4-Dihydroxybenzophenon 131-56-6 352,2′-Dihydroxy-4,4′-dimethoxybenzophenon-5,5′- 3121-60-6disodiumsulfonat 36 Benzoicacid, 2-[4-(diethylamino)-2-hydroxybenzoyl]-,hexylester 302776-68-7 372-(2H-Benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-[1,3,3,3- 155633-54-8tetramethyl-1-[(trimethylsilyl)oxy]disiloxanyl]propyl]phenol 381,1-[(2,2′-Dimethylpropoxy)carbonyl]-4,4-diphenyl-1,3-butadien363602-15-7

In some embodiments, at least one agent is added to any of thecompositions useful in the present invention to stabilize the UVAsunscreen to prevent it from photo-degrading on exposure to UV radiationand thereby maintaining its UVA protection efficacy. A wide range ofcompounds are reported to have these stabilizing properties and shouldbe chosen to complement both the UVA sunscreen and the composition as awhole (See e.g., U.S. Pat. Nos. 5,972,316; 5,968,485; 5,935,556;5,827,508; and WO 00/06110). Preferred examples of stabilizing agentsfor use in the present invention include2-ethylhexyl-2-cyano-3,3-diphenylacrylate,ethyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl-3,3-diphenylacrylate,ethyl-3,3-bis(4-methoxyphenyl)acrylate, diethylhexyl 2,6 napthalate andmixtures thereof (Symrise Chemical Company).

In some preferred embodiments, the present invention provides cosmeticand/or topical dermatological preparations suitable for use as skinprotection creams, cleansing milks, sun screen lotions, nourishingcreams, day creams, night creams etc. In some embodiments, the presentinvention finds use a components of drug (i.e., pharmaceutical)compositions. In additional embodiments, the present invention finds usein decorative cosmetics (e.g., make-up formulations).

In some particularly preferred embodiments, the present inventionprovides sunscreens useful in cosmetic and/or skin care preparations. Inaddition to the active ingredient used according to the embodiments ofthe present invention, in some embodiments, these preparationspreferably additionally comprise at least one broadband filter and/or atleast one UVA filter substance and/or at least one UVB filter substanceand/or at least one inorganic pigment.

In yet further embodiments, the present invention provides personal carecompositions which have UV protection components, but which are notprimarily sunscreens. For example, in some embodiments, UV-A and/or UV-Bfilter substances are incorporated into day creams and/or hair carecompositions.

In additional embodiments, the personal care compositions of the presentinvention comprise cosmetically active ingredients, auxiliaries and/oradditives, as are customarily used in such preparations (e.g.,antioxidants, preservatives, bacteriocides, perfumes, antifoams, dyes,pigments which have a coloring action, thickeners, surface-activesubstances, emulsifiers, emollients, moisturizers and/or humectants,fats, oils, waxes or other customary constituents of a cosmetic ordermatological formulation, such as alcohols, polyols, polymers, foamstabilizers, electrolytes, organic solvents or silicone derivatives).Indeed it is contemplated that various compounds will find use in thevarious embodiments of the present invention, as appropriate for theproduct and the user.

In some embodiments, at least one agent is added to any of thecompositions useful in the present invention to improve the skinsubstantivity of those compositions, particularly to enhance theirresistance to being washed off by water or rubbed off. Examples include,but are not limited to, acrylates/C₁₂₋₂₂ alkylmethacrylate copolymer,acrylate/acrylate copolymer, dimethicone, dimethiconol, graft-copoly(dimethylsiloxane/1-butyl methacrylate), lauryl dimethicone,PVP/Hexadecane copolymer, PVP/Eicosene copolymer, tricontanyl PVP andtrimethoxysiloxysilicate.

In addition to organic sunscreens, in some embodiments, the compositionsof the present invention additionally comprise inorganic physicalsunblocks (See e.g., TFA International Cosmetic Ingredient Dictionary,6^(th) Edition, pp. 1026-28 and 1103 [1995]; Sayre et al., J. Soc.Cosmet. Chem., 41:103-109 [1990]; and Lowe et al., supra). Preferredinorganic physical sunblocks include zinc oxide and titanium dioxide andmixtures thereof.

When used in preferred embodiments, the physical sunblocks are presentin an amount such that the present compositions are transparent on theskin (i.e., non-whitening), preferably from about 0.5% to about 20%,preferably from about 0.5% to about 10%, and more preferably from about0.5% to 5% by weight. When titanium dioxide is used, it can have ananatase, rutile or amorphous structure. Manufacturers of micronizedgrade titanium dioxide and zinc oxide for sunscreen use include, but arenot limited to Tayca Corporation, Uniqema, Shinetsu ChemicalCorporation, Kerr-McGee, Nanophase, Nanosource, Sachtleben, Elementis,and BASF Corporation, as well as their distribution agents and thosecompanies that further process the material for sunscreen use. Physicalsunblock particles (e.g., titanium dioxide and zinc oxide) can beuncoated or coated with a variety of materials including but not limitedto amino acids, aluminum compounds such as alumina, aluminum stearate,aluminum laurate, and the like; carboxylic acids and their salts (e.g.,stearic acid and its salts); phospholipids, such as lecithin; organicsilicone compounds; inorganic silicone compounds such as silica andsilicates and mixtures thereof. In some preferred embodiments, thecompositions of the present invention comprise from about 0.1% to about15%, preferably from about 0.1% to about 7%, and more preferably fromabout 0.5% to about 5%, by weight, of inorganic sunscreen.

In addition to the deleterious effects of some emulsifiers, exposure toother factors is known to harm skin and hair. For example, the harmfuleffect of the ultraviolet portion of solar radiation on the skin isgenerally known. While rays having a wavelength of less than 290 nm(i.e., the UVC region) are absorbed by the ozone layer in the earth'satmosphere, rays in the range between 290 nm and 320 nm (i.e., the UVBregion), cause erythema, simple sunburn or even burns of varyingseverity. The erythema activity maximum of sunlight is given as therelatively narrow region around 308 nm.

Numerous compounds are known to provide protection against harmful UVBradiation. Most commonly, these compounds are derivatives of3-benzylidenecamphor, of 4-aminobenzoic acid, of cinnamic acid, ofsalicylic acid, of benzophenone, and of 2-phenyl-benzimidazole.

It is also important to have available filter substances for the rangebetween about 320 nm and about 400 nm, the UVA region, since its rayscan also cause damage. For a long time it was incorrectly assumed thatthe long-wave UV-A radiation having a wavelength of between 320 nm and400 nm had only a negligible biological action and that, accordingly,the UV-B rays were responsible for most photodamage to the human skin.However, numerous recent studies have shown that UV-A radiation is muchmore harmful than UV-B radiation with regard to the triggering ofphotodynamic, specifically phototoxic, reactions and chronic changes inthe skin. In addition, the harmful effects of UV-B radiation can befurther intensified by exposure to UV-A radiation.

It has been shown that UV-A radiation by itself and under very normaleveryday conditions, is sufficient to damage collagen and elastinfibers, which are of essential importance for the structure and strengthof the skin, within a short period. This leads to chronic light-inducedchanges in the skin, such that the skin prematurely “ages.” The clinicalappearance of skin aged by light typically includes increased wrinklesand lines, and an irregular, furrowed relief. In addition, the skinareas affected by light-induced skin aging often show irregularpigmentation. In some cases, brown patches, keratoses, carcinomas, ormalignant melanomas arise. Skin prematurely aged as a result of everydayUV exposure is also characterized has having lowered activity of theLangerhans cells and slight, chronic inflammation.

Approximately 90% of the ultraviolet radiation which reaches the Earthconsists of UV-A rays. While amount of UV-B radiation reaching Earthvaries widely depending on numerous factors (e.g., time of year and dayand/or degree of latitude), the UV-A radiation levels that reach Earthremain relatively constant on a daily basis, irrespective of the time ofyear and day or geographical factors. Additionally, the majority of UV-Aradiation penetrates the living epidermis, while about 70% of the UV-Brays are retained by the horny layer. Preventive protection against UV-Arays, for example by applying light protection filter substances in theform of a cosmetic or dermatological formulation to the skin, istherefore of fundamental importance.

In general, the light absorption behavior of light protection filtersubstances is very well known and documented, largely due to the factthat most industrialized countries have positive lists for the use ofsuch substances, which impose very strict standards on the documentationthat accompanies each product which incorporates these substances. Forthe concentration of the substances in the finished formulations, theabsorbance values provide a guide, since interaction with substanceswithin the skin or the surface of the skin itself often presentsvariables that may impact how well the compositions perform on eachindividual. However, it is usually difficult to estimate beforehand, howuniformly and thickly the filter substance is distributed in and on thehorny layer of the skin.

To test UV-A protection performance, use is usually made of the IPDmethod (IPD 5 immediate pigment darkening) known to those in the art.This method is similar to the determination of the sun protectionfactor, and provides a method which indicates how much longer skinprotected with the light protection composition can be irradiated withUV-A radiation before the pigmentation which occurs is the same as thatproduced for unprotected skin.

Another test method which has become established throughout Europe isthe Australian standard AS/NZS 2604:1997. In this method, the absorptionof the preparation in the UV-A region is measured. In order to satisfythe standard, the preparation must absorb at least 90% of the UV-Aradiation in the region 320-360 nm.

Of concern in the formulation of sunscreen compositions is that the useconcentration of known light protection filter substances which alsoexhibit high filter action in the UV-A region are often limited by thevery fact that they are combined with other substances which are in theform of solids. Thus, there are certain formulation difficultiesassociate with achieving relatively high sun protection factors and UV-Aprotection performance. However, those of skill in the art are generallyaware of means to overcome and/or compensate for these difficulties.

As light protection filter substances are generally expensive and somelight protection filter substances are additionally difficult toincorporate into cosmetic and/or dermatological preparations inrelatively high concentrations, some embodiments of the presentinvention were designed to provide simple and cost-effectivepreparations which, despite having unusually low concentrations ofconventional UV-A light protection filter substances, neverthelessachieve acceptable or even high UV-A protection performance.

However, as known in the art, UV radiation can also lead tophotochemical reactions which produce products that interfere with theskin's metabolism. These photochemical reaction products arepredominantly free-radical compounds (e.g., hydroxyl radicals).Undefined free-radical photoproducts which form in the skin itself canalso exhibit uncontrolled secondary reactions as a result of their highreactivity. However, singlet oxygen, a non-free-radical excited state ofthe oxygen molecule, can also arise during UV irradiation, as canshort-lived epoxides and many others. Singlet oxygen, for example,differs from normal triplet oxygen (free-radical ground state) by virtueof its increased reactivity. However, excited, reactive “free-radical”triplet states of the oxygen molecule also exist. Thus, in order toprevent these reactions, antioxidants and/or free-radical scavengersfind use in cosmetic and/or dermatological formulations.

The compounds which are commonly used as light protection agents incosmetic and/or dermatological light protection formulations aregenerally characterized as providing good light protection. However,they have the disadvantage that it is sometimes difficult to incorporatethem into the desired formulations in a satisfactory manner.

As indicated above, the sun protection factor (SPF) indicates how muchlonger the skin protected with the light protection composition can beirradiated before the erythema reaction which occurs is the same as forunprotected skin (i.e., ten times as long compared with unprotected skinfor an SPF=10). Consumers are very aware of the meaning of “SPF” andchoose skin and/or hair care products based on the SPF values indicatedon products. Consumers expect to receive reliable information frommanufacturers regarding the sun protection factor, largely due toincreased public awareness of the association between excess sunexposure and skin cancer, as well as premature aging. In addition, insome parts of the world, the degradation of the ozone layer is a majorconcern. Depending upon the skin type and the sun exposure expected,consumers choose products with a lower or a higher SPF. However, thereappears to be a tendency for consumers to select relatively high SPFfactors, particularly for products to be applied to children and thosewith fair skin. In some embodiments, the present invention providescompositions with relatively low concentrations of conventional lightprotection filter substances, yet with SPF values that are acceptable toconsumers.

In some preferred embodiments, the basic constituents of the sunscreenpreparations provided by the present invention include: water or aqueoussolutions; aqueous ethanolic solutions; natural oils and/or chemicallymodified natural oils and/or synthetic oils; fats, waxes and othernatural and synthetic fatty substances, preferably esters of fatty acidswith alcohols of low carbon number (e.g., with isopropanol, propyleneglycol or glycerol), or esters of fatty alcohols with alkanoic acids oflow carbon number or with fatty acids; alcohols, diols or polyols of lowcarbon number, and ethers thereof, preferably ethanol, isopropanol,propylene glycol, glycerol, ethylene glycol, ethylene glycol monoethylor monobutyl ether, propylene glycol monomethyl, monoethyl or monobutylether, diethylene glycol monomethyl or monoethyl ether and analogousproducts. In alternatively preferred embodiments, mixtures of two ormore of these constituents find use in the present invention.

In some preferred embodiments, the composition of the present inventionalso includes preservatives. Such preservatives include, but are notlimited to pentylene glycol, ethylene diamine tetra acetate (EDTA) andtheir salts, chlorhexidine (and its diacetate, dihydrochloride,digluconate derivatives), 1,1,1-trichloro-2-methyl-2-propanol,parachloro metaxylenol, polyhexamethylenebiguanide hydrochloride,dehydroacetic acid, diazolidinyl urea, 2,4-dichlorobenzyl alcohol,4,4-dimethyl-1,3-oxazolidine, formaldehyde (e.g., 37% aqueous solution,with 10-15% methanol to avoid polymerization), glutaraldehyde,dimethylidantoin, imidazolidinyl urea,5-Chloro-2-methyl-4-isothiazolin-3-one, ortho-phenylphenol,4-hydroxybenzoic acid esters (e.g., “paraben”) and its methyl-, ethyl-,propyl-, isopropyl-, butyl-, and isobutyl-esters, trichlosan,2-phenoxyethanol, phenyl mercuric acetate, borate, nitrate,quaternium-15, salicylate, salicylic acid and its salts, calcium,calcium sorbate, sorbic acid and its salts, iodopropanyl butylcarbamatezinc pyrithione, benzyl alcohol, 5-bromo-5-nitro-1,3-dioxane,2-bromo-2-nitropropane-1,3-diol, benzoic acid and its salts, sulfites,bisulfites, phenyoxyethanol, chloroxylenol, diazolidinyl urea,methylparabens, propylparabens, isoproplyparabens, isobutylparabens,butylparabens, ethylparaben, phenoxyethanol PG, and benzalkoniumchloride.

In still further embodiments, preservatives, such as those used in foodand feed applications find use in various compositions of the presentinvention. The following table provides a list of such compounds, aswell as the E number for each compound. However, it is not intended thatthe present invention be limited to these specific preservatives, as itis contemplated that additional preservatives will find use in variousembodiments of the present invention.

Examples of Food Grade Preservatives That Find Use in Embodiments of thePresent Invention 200 Sorbic acid 201 Sodium sorbate 202 Potassiumsorbate 203 Calcium sorbate 210 Benzoic acid 211 Sodium benzoate 212Potassium benzoate 213 Calcium benzoate 214 Ethyl p-hydroxybenzoate 215p-hydroxybenzoic ethyl ester Na salt 216 n-propyl p-hydroxybenzoate 217p-hydroxybenzoic-n-propyl ester Na salt 218 methyl p-hydroxybenzoate 219p-hydroxybenzoic methyl ester Na salt 220 Sulfur dioxide 221 Sodiumsulfite 222 Sodium hydrogensulfite 223 Sodium disulfite 224 Potassiumdisulfite 226 Calcium sulfite 227 Calcium hydrogen sulfite 228 Potassiumhydrogen sulfite 230 Biphenyl (Diphenyl) 231 Orthophenylphenol 232Sodium orthophenylphenoxide 233 Thiabendazole 235 Natamycin 236 Formicacid 237 Sodium formate 238 Calcium formate 239 Hexamethylenetetramine249 Potassium nitrite 250 Sodium nitrite 251 Sodium nitrate 252Potassium nitrate 280 Propionic acid 281 Sodium propionate 282 Calciumpropionate 283 Potassium propionate 290 Carbon dioxide

Additional preservatives that find use in various embodiments includebut are not limited to dibromodicyanobutane(2-bromo-2-bromomethylglutarodinitrile),3-iodo-2-propinylbutylcarbamate, 2-bromo-2-nitropropane-1,3-diol,imidazolidinylurea, 5-chloro-2-methyl-4-isothiazolin-3-one,2-chloroacetamide, benzalkonium chloride, benzyl alcohol, andformaldehyde donors. Further preservatives that find use in variousembodiments of the present invention include phenyl hydroxyalkyl ethers,in particular the compounds known as “phenoxyethanol,” due to theirbactericidal and fungicidal effects on a number of microorganisms.

A variety of optional ingredients such as neutralizing agents, perfumesand perfume solubilizing agents, and coloring agents, also find use insome of the compositions herein. It is preferred that any additionalingredients enhance the skin softness/smoothness benefits of theproduct. In addition it is preferred that any such ingredients do notnegatively impact the aesthetic properties of the product.

Other optional materials include keratolytic agents, as well aswater-soluble and/or solubilizable preservatives preferably at a levelof from about 0.1% to about 5% (e.g., Germall 115, methyl, ethyl, propyland butyl esters of hydroxybenzoic acid, benzyl alcohol, DMDM hydantoiniodopropanyl butylcarbanate available under the trade name Glydant Plusfrom Lonza; EDTA, EUXYL® K400, Bromopol(2-bromo-2-nitropropane-1,3-diol) and phenoxypropanol); anti-bacterials(e.g., IRGASAN®) and phenoxyethanol (preferably at levels of from about0.1% to about 5%); as well as soluble or colloidally-solublemoisturizing agents such as hyaluronic acid, chondroitin sulfate, andstarch-grafted sodium polyacrylates (e.g., SANWET® IM-1000, IM-1500 andIM-2500, available from Celanese Superabsorbent Materials, Portsmith,Va., See e.g., U.S. Pat. No. 4,076,663; vitamins such as vitamin A,vitamin C, vitamin E and derivatives thereof and building blocks thereofsuch as phytantriol, and vitamin K and components thereof such as thefatty alcohol dodecatrienol; alpha and beta hydroxyacids; aloe vera;sphingosines and phytosphingosines, cholesterol; skin whitening agents;N-acetyl cysteine; colouring agents; antibacterial agents such asTCC/TCS, also known as triclosan and trichlorocarbon; perfumes andperfume solubilizers. Examples of alpha hydroxy acids include glycolicacid, lactic acid, malic acid, citric acid, glycolic acid in conjunctionwith ammonium glycolate, alpha-hydroxy ethanoic acid,alpha-hydroxyoctanoic acid, alpha-hydroxycaprylic acid, hydroxycaprylicacid, mixed fruit acid, tri-alpha hydroxy fruit acids, triple fruitacid, sugar cane extract, alpha hydroxy and botanicals, I-alpha hydroxyacid and glycomer in crosslinked fatty acids (e.g., alpha nutrium).Preferred examples of alpha hydroxy acids are glycolic acid and lacticacid. It is preferred that alpha hydroxy acids are used in levels of upto about 10%. It is not intended that the present invention be limitedto any particular compound derived from any particular source, as anysuitable additive compound, whether obtained from natural sources orthrough synthesis in the laboratory find use in the present invention.

Other optional materials include water-soluble or solubilizablepreservatives preferably at a level of from about 0.1% to about 5% each,such as Germall 115, methyl, ethyl, propyl and butyl esters ofhydroxybenzoic acid, benzyl alcohol, DMDM hydantoin iodopropanylbutylcarbanate available under the trade name Glydant Plus from Lonza,EDTA, Euxyl® K400, Bromopol (2-bromo-2-nitropropane-1,3-diol), pentyleneglycol and phenoxypropanol; anti-bacterials such as Irgasan® andphenoxyethanol (preferably at levels of from 0.1% to about 5%).Antibacterial agents such as TCC/TCS, also known as triclosan andtrichlorocarbon are also useful in compositions of the presentinvention.

Yet other antimicrobial agents are likewise suitable for use in variousembodiments of the present invention, including but not limited to2,4,4′-trichloro-2′-hydroxydiphenyl ether (i.e., IRGASAN®),1,6-di(4-chlorophenylbiguanido)hexane (i.e., CHLORHEXIDIN),3,4,4′-trichlorocarbanilide, quaternary ammonium compounds, oil ofcloves, mint oil, thyme oil, triethyl citrate, FARNESOL®(3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) and the active ingredientsand/or active ingredient combinations described in DE-37 40 186, DE-3938 140, DE-42 04 321, DE-42 29 707, DE-43 09 372, DE-44 11 664, DE-1 9541 967, DE-1 95 43 695, DE-1 95 43 696, DE-1 95 47 160, DE-1 96 02 108,DE-196 02 110, DE-196 02 111, DE-196 31 003, DE-196 31 004, DE-196 34019, DE-42 29 737, DE-42 37 081, DE-43 24 219, DE-44 29 467, DE-44 23410, and DE-195 16 705, all of which are hereby incorporated byreference. In still further embodiments, sodium hydrogencarbonate isalso included in some compositions of the present invention. However, itis not intended that the present invention be limited to any particularantimicrobial nor combination of anti-microbial, as various compoundshaving such effects will find use in various embodiments of the presentinvention.

In additional embodiments of the personal care compositions of thepresent invention, compounds such as anti-irritants and/oranti-inflammatory actives are included. In some embodiments, batylalcohol (a-octadecyl glyceryl ether), selachyl alcohol (a-9-octadecenylglyceryl ether), chimyl alcohol (a-hexadecyl glyceryl ether), bisabolol,and/or panthenol are included. However, it is not intended that thepresent invention be limited to the incorporation of any specificanti-irritant(s) and/or anti-inflammatory(ies), as various compoundssuitable for such applications find use in the present invention.

Neutralizing agents suitable for use in neutralizing acidic groupcontaining hydrophilic gelling agents herein include sodium hydroxide,potassium hydroxide, ammonium hydroxide, monoethanolamine,diethanolamine, amino methyl propanol, tris-buffer and triethanolamine.

Other optional materials that find use in the present invention includeany of the numerous functional and/or active ingredients known to thoseskilled in the art (See e.g., McCutcheon's Functional Materials, NorthAmerican and International Editions, MC Publishing Co. [2003]) Asindicated above, non-limiting examples include keratolytic agents;soluble or colloidally-soluble moisturizing agents such as hyaluronicacid and chondroitin sulfate; vitamins such as vitamin A, vitamin C,vitamin E, vitamin K and derivatives thereof and building blocksthereof; phytantriol; fatty alcohols such as dodecatrienol; alpha andbeta hydroxyacids; aloe vera; sphingosines and phytosphingosines,cholesterol; skin whitening agents; N-acetyl cysteine; coloring agents;Examples of alpha hydroxy acids include glycolic acid, lactic acid,malic acid, and citric acid (whether derived synthetically or fromnatural sources and whether used alone or in combination) and theiresters or relevant buffered combinations. Other examples ofalpha-hydroxy acids include: alpha-hydroxy ethanoic acid,alpha-hydroxyoctanoic acid, alpha-hydroxycaprylic acid, andhydroxycaprylic acid. Preferred examples of alpha hydroxy acids includeglycolic acid and lactic acid. It is preferred that alpha hydroxy acidsare used in levels of up to about 10%.

Optional materials include pigments that, where water-insoluble,contribute to and are included in the total level of oil phaseingredients. Pigments suitable for use in the compositions of thepresent invention can be organic and/or inorganic. Also included withinthe term “pigment” are materials having a low color or luster, such asmatte finishing agents, light scattering agents, and formulation aidssuch as micas, seracites, and carbonate salts. Further examples ofsuitable pigments include titanium dioxide, iron oxides, glutamate ironoxides, zinc oxide, bismuth oxychloride, ultramarine blue (all of whichmay be either pre-dispersed and/or pre-coated or not) D&C dyes andlakes, FD&C colors, natural color additives such as carmine, andmixtures thereof. Depending upon the type of composition, a mixture ofpigments is usually used in preferred embodiments of the presentinvention. Preferred pigments for use herein from the viewpoint ofmoisturization, skin feel, skin appearance and emulsion compatibilityare treated pigments. In some embodiments, the pigments are treated withcompounds, including but not limited to amino acids, silicones, lecithinand ester oils.

In some embodiments, the present invention provides compositionscomprising pigments, including, but not limited to inorganic pigmentsbased on metaloxides and/or other in water slightly soluble or insolublemetal compounds such as zinc oxides (ZnO), titanium (TiO₂), iron (e.g.,Fe₂O₃), zirconium (ZrO₂), silica (SiO₂), manganese (e.g., MnO),aluminium (Al₂O₃), cer (e.g., Ce₂O₃), and mixed compositions of theseoxides, as well as blends thereof. In some preferred embodiments, themetaloxides are microfine, while in alternative preferred embodiments,the metaloxides are pigment grade. In yet additional embodiments, thepigments are “coated” such that they are surface treated. In somepreferred embodiments, the coating comprises a thin, hydrophobic filmlayer, while in other embodiments, the coating comprises a thin,hydrophilic film layer.

As used herein, the terms “pigment,” “color pigment,” and “dye” used inreference to the compositions of the present invention encompasses anycompound that provides a color to the composition and/or imparts a colorto the surface (e.g., skin and/or hair) to which the composition isapplied. In some embodiments, the dyes and pigments are chosen from thelist of cosmetic colorants provided by the Cosmetics Directive or theEC. In most cases, these dyes and pigments are identical to the dyesapproved for foods. Preferred pigments/dyes include for example,titanium dioxide, mica, iron oxides (e.g., Fe₂O₃, Fe₃O₄, FeO(OH)) and/ortin oxide. Advantageous pigments/dyes include for example, carmine,Berlin blue, chrome oxide green, ultramarine blue and/or manganeseviolet. In some preferred embodiments, the pigments/dyes include, butare not limited to those in the following table. The Colour IndexNumbers (CIN) those known in the art (See, Society of Dyers andColourists, Rowe Colour Index, 3rd Edition, Bradford, England, [1971]).

CHEMICAL OR OTHER NAME CIN COLOR Pigment Green 10006 green Acid Green 110020 green 2,4-Dinitrohydroxynaphthalene-7-sulfonic acid 10316 yellowPigment Yellow 1 11680 yellow Pigment Yellow 3 11710 yellow PigmentOrange 1 11725 orange 2,4-Dihydroxyazobenzene 11920 orange Solvent Red 312010 red 1-(2′-Chloro-4′-nitro-1′-phenylazo)-2-hydroxy- 12085 rednaphthalene Pigment Red 3 12120 red Ceres red; Sudan red; Fat Red G12150 red Pigment Red 112 12370 red Pigment Red 7 12420 red PigmentBrown 1 12480 brown 4-(2′-Methoxy-5′-sulfodiethylamido-1′-phenylazo)-3-12490 red hydroxy-5″-chloro-2″,4″-dimethoxy-2-naphthanilide DisperseYellow 16 12700 yellow 1-(4-Sulfo-1-phenylazo)-4-aminobenzene-5-sulfonic13015 yellow acid 2,4-Dihydroxyazobenzene-4′-sulfonic acid 14270 orange2-(2,4-Dimethylphenylazo-5-sulfo)-1-hydroxy- 14700 rednaphthalene-4-sulfonic acid2-(4-Sulfo-1-naphthylazo)-1-naphthol-4-sulfonic acid 14720 red2-(6-Sulfo-2,4-xylylazo)-1-naphthol-5-sulfonic acid 14815 red1-(4′-Sulfophenylazo)-2-hydroxynaphthalene 15510 orange1-(2-Sulfo-4-chloro-5-carboxy-1-phenylazo)-2- 15525 redhydroxynaphthalene 1-(3-Methylphenylazo-4-sulfo)-2-hydroxynaphthalene15580 red 1-(4′,(8′)-Sulfonaphthylazo)-2-hydroxynaphthalene 15620 red2-Hydroxy-1,2′-azonaphthalene-1′-sulfonic acid 15630 red3-Hydroxy-4-phenylazo-2-naphthylcarboxylic acid 15800 red1-(2-Sulfo-4-methyl-1-phenylazo)-2-naphthyl- 15850 red carboxylic acid1-(2-Sulfo-4-methyl-5-chloro-1-phenylazo)-2- 15865 redhydroxynaphthalene-3-carboxylic acid1-(2-Sulfo-1-naphthylazo)-2-hydroxynaphthalene-3- 15880 red carboxylicacid 1-(3-Sulfo-1-phenylazo)-2-naphthol-6-sulfonic acid 15980 orange1-(4-Sulfo-1-phenylazo)-2-naphthol-6-sulfonic acid 15985 yellow AlluraRed 16035 red 1-(4-Sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic 16185red acid Acid Orange 10 16230 orange1-(4-Sulfo-1-naphthylazo)-2-naphthol-6,8-disulfonic 16255 red acid1-(4-Sulfo-1-naphthylazo)-2-naphthol-3,6,8-trisulfonic 16290 red acid8-Amino-2-phenylazo-1-naphthol-3,6-disulfonic acid 17200 red Acid Red 118050 red Acid Red 155 18130 red Acid Yellow 121 18690 yellow Acid Red180 18736 red Acid Yellow 11 18820 yellow Acid Yellow 17 18965 yellow4-(4-Sulfo-1-phenylazo)-1-(4-sulfophenyl)-5-hydroxy- 19140 yellowpyrazolone-3-carboxylic acid Pigment Yellow 16 20040 yellow2,6-(4′-Sulfo-2″,4″-dimethyl)bisphenylazo)-1,3- 20170 orangedihydroxybenzene Acid Black 1 20470 black Pigment Yellow 13 21100 yellowPigment Yellow 83 21108 yellow Solvent Yellow 21230 yellow Acid Red 16324790 red Acid Red 73 27290 red2-[4′-(4″-Sulfo-1″-phenylazo)-7′-sulfo-1′-naphthylazo]- 27755 black1-hydroxy-7-aminonaphthalene-3,6-di-sulfonic acid4′-[(4″-Sulfo-1″-phenylazo)-7′-sulfo-1′-naphthylazo]-1- 28440 blackhydroxy-8-acetylaminonaphthalene-3,5-disulfonic acid Direct Orange 34,39, 44, 46, 60 40215 orange Food Yellow 40800 orangetrans-β-Apo-8′-carotinaldehyde (C30) 40820 orange trans-Apo-8′-carotinicacid (C30)-ethyl ester 40825 orange Canthaxanthin 40850 orange Acid Blue1 42045 blue 2,4-Disulfo-5-hydroxy-4′-4″-bis(diethylamino)tri- 42051blue phenylcarbinol 4-[(4-N-Ethyl-p-sulfobenzylamino)phenyl(4-hydroxy-42053 green 2-sulfophenyl)(methylene)-1-(N-ethyl-N-p-sulfobenzyl)-2,5-cyclohexadienimine] Acid Blue 7 42080 blue(N-Ethyl-p-sulfobenzylamino)phenyl(2- 42090 bluesulfophenyl)methylene-(N-ethyl-N-p- sulfobenzyl)D2,5-cyclohexadienimineAcid Green 9 42100 greenDiethyldisulfobenzyldi-4-amino-2-chloro-di-2-methyl- 42170 greenfuchsonimmonium Basic Violet 14 42510 violet Basic Violet 2 42520 violet2′-Methyl-4′-(N-ethyl-N-m-sulfobenzyl)amino-4″-(N- 42735 bluediethyl)amino-2-methyl-N-ethyl-N-m- sulfobenzylfuchsonimmonium4′-(N-Dimethyl)amino-4″-(N-phenyl)aminonaphtho-N- 44045 bluedimethyl-fuchsonimmonium2-Hydroxy-3,6-disulfo-4,4′-bisdimethylaminonaphtho- 44090 greenfuchsonimmonium Acid Red 52 45100 red3-(2′-Methylphenylamino)-6-(2′-methyl-4′-sulfophenyl- 45190 violetamino)-9-(2″-carboxyphenyl)xanthenium salt Acid Red 50 45220 redPhenyl-2-oxyfluorone-2-carboxylic acid 45350 yellow4,5-Dibromofluorescein 45370 orange 2,4,5,7-Tetrabromofluorescein 45380red Solvent Dye 45396 orange Acid Red 98 45405 red3′,4′,5′,6′-Tetrachloro-2,4,5,7-tetrabromofluorescein 45410 red4,5-Diiodofluorescein 45425 red 2,4,5,7-Tetraiodofluorescein 45430 redQuinophthalone 47000 yellow Quinophthalonedisulfonic acid 47005 yellowAcid Violet 50 50325 violet Acid Black 2 50420 black Pigment Violet 2351319 violet 1,2-Dioxyanthraquinone, calcium-aluminum complex 58000 red3-Oxypyrene-5,8,10-sulfonic acid 59040 green1-Hydroxy-4-N-phenylaminoanthraquinone 60724 violet1-Hydroxy-4-(4′-methylphenylamino)anthraquinone 60725 violet Acid Violet23 60730 violet 1,4-Di(4′-methylphenylamino)anthraquinone 61565 green1,4-Bis(o-sulfo-p-toluidino)anthraquinone 61570 green Acid Blue 80 61585blue Acid Blue 62 62045 blue N,N′-Dihydro-1,2,1′,2′-anthraquinone azine69800 blue Vat Blue 6; Pigment Blue 64 69825 blue Vat Orange 7 71105orange Indigo 73000 blue Indigo-disulfonic acid 73015 blue4,4′-Dimethyl-6,6′-dichlorothioindigo 73360 red5,5′-Dichloro-7,7′-dimethylthioindigo 73385 violet Quinacridone Violet19 73900 violet Pigment Red 122 73915 red Pigment Blue 16 74100 bluePhthalocyanine 74160 blue Direct Blue 86 74180 blue ChlorinatedPhthalocyanines 74260 green Natural Yellow 6, 19; Natural Red 1 75100yellow Bixin, Nor-Bixin 75120 orange Lycopene 75125 yellow trans-alpha-,beta- and gamma-carotene 75130 orange Keto- and/or hydroxyl derivates ofcarotene 75135 yellow Guanine or pearlizing agent 75170 white1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene- 75300 yellow3,5-dione Complex salt (Na, Al, Ca) of carminic acid 75470 redChlorophyll a and b; copper compounds of 75810 green chlorophylls andChlorophyllins Aluminum 77000 white Hydrated alumina 77002 white Hydrousaluminum silicates 77004 white Ultramarine 77007 blue Pigment Red 101und 102 77015 red Barium sulfate 77120 white Bismuth oxychloride and itsmixtures with mica 77163 white Calcium carbonate 77220 white Calciumsulfate 77231 white Carbon 77266 black Pigment Black 9 77267 black Carbomedicinalis vegetabilis 77268 black Chromium oxide 77288 green Chromiumoxide, hydrous 77289 green Pigment Blue 28, Pigment Green 14 77346 greenPigment Metal 2 77400 brown Gold 77480 brown Iron oxides and hydroxides77489 orange Iron oxide 77491 red Iron oxide, hydrated 77492 yellow Ironoxide 77499 black Mixtures of iron (II) and iron(III)hexacyano-ferrate77510 blue Pigment White 18 77713 white Manganese ammonium diphosphate77742 violet Manganese phosphate; Mn3(PO4)2 □ 7 H20 77745 red Silver77820 white Titanium dioxide and its mixtures with mica 77891 white Zincoxide 77947 white 6,7-Dimethyl-9-(1′-D-ribityl)-isoalloxazine, yellowlactoflavine Sugar coloring brown Capsanthin, capsorubin orange Betaninred Benzopyrylium salts, Anthocyans red Aluminum, zinc, magnesium andcalcium stearate white Bromothymol blue blue Bromocresol green greenAcid Red 195 red

In yet further embodiments, compositions of the present inventionfurther comprise one or more substances from the following group:2,4-dihydroxyazobenzene,1-(2′-chloro-4′-nitro-1′-phenylazo)-2-hydroxynaphthalene, Ceres Red,2-(4-sulfo-1-naphthylazo)-1-naphthol-4-sulfonic acid, calcium salt of2-hydroxy-1,2′-azonaphthalene-1′-sulfonic acid, calcium and barium saltsof 1-(2-sulfo-4-methyl-1-phenylazo)-2-naphthylcarboxylic acid, calciumsalt of 1-(2-sulfo-1-naphthylazo)-2-hydroxynaphthalene-3-carboxylicacid, aluminum salt of 1-(4-sulfo-1-phenylazo)-2-naphthol-6-sulfonicacid, aluminum salt of1-(4-sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic acid,1-(4-sulfo-1-naphthylazo)-2-naphthol-6,8-disulfonic acid, aluminum saltof4-(4-sulfo-1-phenylazo)-1-(4-sulfophenyl)-5-hydroxypyrazolone-3-carboxylicacid, aluminum and zirconium salts of 4,5-dibromofluorescein, aluminumand zirconium salts of 2,4,5,7-tetrabromofluorescein,3′,4′,5′,6′-tetrachloro-2,4,5,7-tetrabromofluorescein and its aluminumsalt, aluminum salt of 2,4,5,7-tetraiodofluorescein, aluminum salt ofquinophthalone disulfonic acid, aluminum salt of indigo disulfonic acid,red and black iron oxide (CIN: 77 491 (red) and 77 499 (black)), ironoxide hydrate (CIN: 77 492), manganese ammonium diphosphate and titaniumdioxide.

In yet further embodiments, oil-soluble natural dyes, such as, forexample, paprika extracts, β-carotene or cochenille find use in thepresent invention.

In yet additional embodiments, gel cream compositions of the presentinvention comprise pearlescent pigments. In some preferred embodiments,various pearlescent pigments find use in the present invention,including but not limited to “natural pearlescent pigments” (e.g.,“pearl essence” [guanine/hypoxanthine mixed crystals from fish scales],“mother of pearl” [ground mussel shells]), and “monocrystallinepearlescent pigments” (e.g., bismuth oxychloride [BiOCl]); and “layersubstrate pigments” (e.g. mica/metal oxide).

Bases for pearlescent pigments include, but are not limited topulverulent pigments, castor oil dispersions of bismuth oxychlorideand/or titanium dioxide, bismuth oxychloride and/or titanium dioxide onmica. The luster pigment listed under CIN 77163, for example, isparticularly advantageous.

An additional group of pearlescent pigments based on mica/metal oxidefind particular use in the present invention is provided below.

COATING/LAYER GROUP THICKNESS COLOR Silver-white pearlescent pigmentsTiO2: 40-60 nm silver Interference pigments TiO2: 60-80 nm yellow TiO2:80-100 nm red TiO2: 100-140 nm blue TiO2: 120-160 nm green Color lusterpigments Fe2O3 bronze Fe2O3 copper Fe2O3 red Fe2O3 red-violet Fe2O3red-green Fe2O3 black Combination pigments TiO2/Fe2O3 gold shadesTiO2/Cr2O3 green TiO2/Berlin blue deep blue TiO2/carmine red

In some preferred embodiments, the pearlescent pigments available fromMerck under the trade names Timiron, Colorona or Dichrona find use inthe present invention. However, it is not intended that the presentinvention be limited to the specific pigments listed herein. Indeed,pearlescent pigments that find use in the present invention areobtainable from numerous sources. For example, other substrates apartfrom mica can be coated with further metal oxides, such as, for example,silica and the like. SiO₂ particles coated with, for example, TiO₂ andFe₂O₃ (“ronaspheres”), which are sold by Merck and are particularlysuitable for the optical reduction of fine lines find use in the presentinvention.

In alternative embodiments, the substrate (e.g., mica) is not included.In some preferred embodiments, particular preference is given topearlescent pigments prepared using SiO₂. Such pigments, which may alsoadditionally have goniochromatic effects, are available, for example,under the trade name Sicopearl Fantastico, available from BASF.

In additional embodiments, pigments obtained from Engelhard/Mearl basedon calcium sodium borosilicate which have been coated with titaniumdioxide also find use. These are available under the name Reflecks. Inaddition to the color, as a result of their particle size of from 40 nmto 180 mm, they have a glitter effect.

In yet further embodiments, effect pigments which are available underthe trade name Metasomes Standard/Glitter in various colors (yellow,red, green, blue) from Flora Tech find use in the compositions of thepresent invention. The glitter particles are present here in themixtures with various auxiliaries and dyes (such as, for example, thedyes with the Colour Index (CI) Numbers 19140, 77007, 77289, 77491).

In some embodiments, the dyes and pigments are present eitherindividually or in a mixture. In alternative embodiments, they aremutually coated with one another, different coating thicknessesgenerally giving rise to different color effects. In some embodiments,the total amount of dyes and color-imparting pigments is chosen from arange of concentrations (e.g., from about 0.1% by weight to about 30% byweight; preferably from about 0.5 to about 15% by weight; and mostpreferably from about 1.0 to about 10% by weight, in each case based onthe total weight of the preparations).

In preferred embodiments, the pH of the compositions herein is in therange from about 3.5 to about 10, preferably from about 4 to about 8,and more preferably from about 5 to about 7, wherein the pH of the finalcomposition is adjusted by addition of acidic, basic or buffer salts asnecessary, depending upon the composition of the forms and thepH-requirements of the compounds.

The compositions of the present invention are prepared by standardtechniques well known to those skilled in the art. In general theaqueous phase and/or the oil phase are prepared separately, withmaterials of similar phase partitioning being added in any order. If thefinal product is an emulsion, the two phases are then combined withvigorous stirring and/or homogenization as necessary, to reduce the sizeof the internal phase droplets. Any ingredients in the formulation withhigh volatility, or which are susceptible to hydrolysis or decompositionat high temperatures, are added with gentle stirring towards the end ofthe process, post emulsification if applicable. Dosage frequency andamount will depend upon the desired performance criteria.

In some embodiments of the present invention, method of decreasing VEGFactivity are provided. In these embodiments, the methods compriseapplying to an organism in need thereof an effective amount of any oneof the compounds set forth herein. In additional preferred embodiments,the present invention provides compounds for treatment of an organism inneed thereof, including humans and other animals.

In some still further embodiments, the present invention comprises atleast one creatine and/or creatine derivative. Creatine has thefollowing structure:

In some preferred embodiments of the personal care compositions of thepresent invention creatine phosphate, creatine sulfate, creatineacetate, creatine ascorbate, and/or derivatives esterified at thecarboxyl group with mono- or polyfunctional alcohols find use.

In some additional embodiments, the personal care compositions of thepresent invention contain L-carnitine[3-hydroxy-4-(trimethylammonio)butyrobetaine]. Acylcarnitines have thefollowing general structure:

where R is chosen from the group of branched and unbranched alkylradicals having up to 10 carbon atoms, and find use in some embodimentsof the present invention. In some preferred embodiments,propionylcarnitine and/or acetylcarnitine find use. Both enantiomers (Dand L form), as well as mixtures and racemates of the D- and L-formsfind use in some personal care compositions of the present invention.

In some further embodiments, the active ingredients of the presentinvention include, but are not limited to sericoside, pyridoxol, vitaminK, biotin, and aroma substances. In addition, it is not intended thatthe active ingredients present in the personal care compositions of thepresent invention be limited to any particular constituent and/ormixture(s) of actives. Indeed, it is intended that various actives andmixtures of actives will find use in various embodiments of the presentinvention. It is also not intended that the concentration(s) of suchactives be limited to any particular level. In some embodiments, theconcentration is from about 0.001 to about 30% by weight, while in otherembodiments it is from about 0.05 to about 20% by weight, and in stillfurther embodiments, it is from about 0.1 to about 10% by weight, basedon the total weight of the preparation. It is further contemplated thatthose of skill in the art will formulate personal care compositions ofthe present invention with active(s) concentrations that are suitablefor the intended use of the compositions.

The yet further embodiments, the present invention provides methods forthe preparation of the compositions of the present invention. In someembodiments, these methods include combining and heating theconstituents of the oil phase and/or the water phase separately, andthen combining them together with stirring. In some preferredembodiments, the phases are homogenized. In some particularly preferredembodiments, the compositions are stirred with moderate to high input ofenergy, advantageously using a gear rim dispersing machine at a rotarynumber up to at most 10000 rpm (preferably in the range from about 2500to about 7700 rpm).

6.2 EXPERIMENTAL

The present invention is described in further detail in the followingExamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.All references cited are herein specifically incorporated by referencefor all that is described therein. The following examples are offered toillustrate, but not to limit the claimed invention

In the experimental disclosure which follows, the followingabbreviations apply PI (proteinase inhibitor), BBI (Bowman-BirkInhibitor from Glycine max Acc. No. P01055), BBI-AV (Bowman-BirkInhibitor Anti-VegF), STI (Soybean Trypsin inhibitor from Glycine max);VEGF and VegF (vascular endothelial growth factor); BBdb (Bowman BirkInhibitor from Dolichos biflorus Acc. No. AAK97765), BBsb3 (Bowman BirkInhibitor from Glycine max (soybean) protease inhibitor IV or D-II), andBBtc (Bowman Birk Inhibitor from Torresea cearensis), FGF-5 (fibroblastgrowth factor 5), TGFβ (Transforming growth factor β), TNFα (Tumornecrosis factor α), ppm (parts per million); M (molar); mM (millimolar);μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); μm (grams); mg (milligrams); μg(micrograms); pg (picograms); L (liters); ml and mL (milliliters); μland μL (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); U (units); V (volts); MW (molecularweight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s)(hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND(not done); NA (not applicable); rpm (revolutions per minute); H₂O(water); dH₂O (deionized water); (HCl (hydrochloric acid); aa (aminoacid); bp (base pair); kb (kilobase pair); kD (kilodaltons); cDNA (copyor complimentary DNA); DNA (deoxyribonucleic acid); ssDNA (singlestranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); MgCl₂ (magnesium chloride); NaCl(sodium chloride); w/v (weight to volume); v/v (volume to volume); g(gravity); OD (optical density); Dulbecco's phosphate buffered solution(DPBS); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl,2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol,2.31 g/l KH₂PO₄, and 12.54 g/l K₂HPO₄); OD₂₈₀ (optical density at 280nm); OD₆₀₀ (optical density at 600 nm); A₄₀₅ (absorbance at 405 nm);Vmax (the maximum initial velocity of an enzyme catalyzed reaction);PAGE (polyacrylamide gel electrophoresis); PBS (phosphate bufferedsaline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PBST(PBS+0.25% TWEEN® 20); PEG (polyethylene glycol); PCR (polymerase chainreaction); RT-PCR (reverse transcription PCR); SDS (sodium dodecylsulfate); Tris (tris(hydroxymethyl)aminomethane); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); SDS (sodium dodecylsulfate); bME, BME and βME(beta-mercaptoethanol or 2-mercaptoethanol); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Tricine(N-[tris-(hydroxymethyl)-methyl]-glycine); CHES (2-(N-cyclo-hexylamino)ethane-sulfonic acid); TAPS(3-{[tris-(hydroxymethyl)-methyl]-amino}-propanesulfonic acid); CAPS(3-(cyclo-hexylamino)-propane-sulfonic acid; DMSO (dimethyl sulfoxide);DTT (1,4-dithio-DL-threitol); Glut and GSH (reduced glutathione); GSSG(oxidized glutathione); TCEP (Tris[2-carboxyethyl]phosphine); Ci(Curies) mCi (milliCuries); μCi (microCuries); TLC (thin layerachromatography); Ts (tosyl); Bn (benzyl); Ph (phenyl); Ms (mesyl); Et(ethyl), Me (methyl); Taq (Thermus aquaticus DNA polymerase); Klenow(DNA polymerase I large (Klenow) fragment); rpm (revolutions perminute); EGTA (ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); bla(β-lactamase or ampicillin-resistance gene); GE Healthcare (GEHealthcare, Chalfont St. Giles, United Kingdom); DNA2.0 (DNA2.0, MenloPark, Calif.); OXOID (Oxoid, Basingstoke, Hampshire, UK); Megazyme(Megazyme International Ireland Ltd., Bray Business Park, Bray, Co.,Wicklow, Ireland); Corning (Corning Life Sciences, Corning, N.Y.); (NEN(NEN Life Science Products, Boston, Mass.); Pharma AS (Pharma AS, Oslo,Norway); Dynal (Dynal, Oslo, Norway); Bio-Synthesis (Bio-Synthesis,Lewisville, Tex.); ATCC (American Type Culture Collection, Rockville,Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma ChemicalCo., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Pisacataway, N.J.);NCBI (National Center for Biotechnology Information); Applied Biosystems(Applied Biosystems, Foster City, Calif.); Clontech (CLONTECHLaboratories, Palo Alto, Calif.); Operon Technologies (OperonTechnologies, Inc., Alameda, Calif.); MWG Biotech (MWG Biotech, HighPoint, N.C.); Oligos Etc (Oligos Etc. Inc, Wilsonville, Oreg.); Bachem(Bachem Bioscience, Inc., King of Prussia, Pa.); Difco (DifcoLaboratories, Detroit, Mich.); Mediatech (Mediatech, Herndon, Va.; SantaCruz (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.); BioVeris(BioVeris Corp., Gaithersburg, Md.); Oxoid (Oxoid Inc., Ogdensburg,N.Y.); Worthington (Worthington Biochemical Corp., Freehold, N.J.);GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.);Millipore (Millipore, Billerica, Mass.); Bio-Rad (Bio-Rad, Hercules,Calif.); Invitrogen (Invitrogen Corp., San Diego, Calif.); NEB (NewEngland Biolabs, Beverly, Mass.); Sigma (Sigma Chemical Co., St. Louis,Mo.); Pierce (Pierce Biotechnology, Rockford, Ill.); Takara (Takara BioInc. Otsu, Japan); Roche (Hoffmann-La Roche, Basel, Switzerland); EMScience (EM Science, Gibbstown, N.J.); Qiagen (Qiagen, Inc., Valencia,Calif.); Biodesign (Biodesign Intl., Saco, Me.); Aptagen (Aptagen, Inc.,Herndon, Va.); Molecular Devices (Molecular Devices, Corp., Sunnyvale,Calif.); R&D Systems (R&D Systems, Minneapolis, Minn.); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); Marsh (MarshBiosciences, Rochester, N.Y.); Bio-Tek (Bio-Tek Instruments, Winooski,Vt.); (Biacore (Biacore, Inc., Piscataway, N.J.); PeproTech (PeproTech,Rocky Hill, N.J.); SynPep (SynPep, Dublin, Calif.); and Microsoft(Microsoft, Inc., Redmond, Wash.).

Example 1 Production of BCE103-BBI Fusion Proteins in B. subtilis

In this Example, experiments conducted to produce BCE103-BBI fusionproteins in B. subtilis are described. The DNA sequence of the syntheticgene (Operon Technologies) coding for the pro-BBI protein with aC-terminal hexa-histidine tag used in these experiments is:

(SEQ ID NO: 10) AACCTGCGTCTGTCTAAGCTTGGCCTGCTTATGAAATCAGACCATCAGCACAGCAATGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATAGCTGTCATAGTGCATGCAAAAGCTGTATCTGCGCCCTGAGTTATCCAGCTCAATGTTTTTGCGTCGACATCACGGACTTCTGCTATGAGCCATGTAAACCAAGCGAGGACGATAAAGAGAACCATCATCACCATCACCAT

The protein sequence of pro-BBI with a C-terminal hexa-histidine taggedcoded for by the above synthetic gene is:

(SEQ ID NO: 11) NLRLSKLGLLMKSDHQHSNDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKENHHHHHH

The portion of the DNA sequence of the synthetic gene that codes for themajor mature form of BBI is:

(SEQ ID NO: 12) GACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATAGCTGTCATAGTGCATGCAAAAGCTGTATCTGCGCCCTGAGTTATCCAGCTCAATGTTTTTGCGTCGACATCACGGACTTCTGCTATGAGCCATGTAAACCAAGCGAGGA CGATAAAGAGAAC

The protein sequence of the major mature form of BBI coded by the abovesynthetic gene is:

(SEQ ID NO: 13) DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKEN

The PCR primers used to amplify the BBI gene for fusion to the BCE103cellulase expression cassette in the pJ103 vector were:

BBIfusion_FW: (SEQ ID NO: 14) 5′ CAGCACGGATCCAGACGATGAGAGCTCTAAACCC 3′BBIHindIII_RV: (SEQ ID NO: 15)5′ CTGCAGAAGCTTAAAAATAAAAAAACGGATTTCCTTCAGGAAATCCGTCCTCTGTTAACTTTTAGTTCTCTTTATCGTCCTCGC 3′ BBIHIS-HindIII_RV: (SEQ ID NO:16) 5′ CTGCAGAAGCTTAAAAATAAAAAAACGGATTTCCTTCAGGAAATCCGTCCTCTGTTAACTTTTAATGGTGATGGTGATGATGGTTCTC 3′

The sequence of the aprE-BCE103-BBI-HisTag expression cassette(EcoRI-HindIII) that was cloned into the pJM103 integration vector isprovided in FIG. 1. A schematic plasmid map of the pJM103BBIHisexpression vector is provided in FIG. 2.

The alkaline cellulase (BCE103) gene (See, van Soligen, U.S. Pat. No.6,063,611, hereby incorporated by reference) fused to the B. subtilisaprE promoter and signal sequence, was cloned from pUCAPR103 (Shaw etal., J. Mol. Biol., 320:303-309 [2002]) as an EcoRI-BamHI fragment(i.e., a fragment that carries the coding sequence of the BCE103catalytic domain and first cellulose binding domain linker only) intopJM103 (Perego, “Integrational vectors for genetic manipulation inBacillus subtilis” In, Bacillus subtilis and Other Gram-positiveBacteria: Biochemistry, Physiology, and Molecular Genetics, Sonenshein,Hoch, and Losick (eds), American Society for Microbiology, WashingtonD.C., pp. 615-624 [1993]). A gene encoding the soybean Bowman-Birkprotease inhibitor (BBI) (Swiss-Prot Accession #P01055; See, Odani andIkenaka, J. Biochem., 71: 839-848 [1972]) with a C-terminalhexa-histidine tag (His-Tag) was synthesized by Operon Technologies(See, DNA sequence above). The BBI gene was amplified by PCR withprimers (all primers were synthesized by MWG Biotech, Oligos Etc., orOperon Technologies) that generated a 5′ BamHI site in the correctreading frame with the BCE103 gene, and at the 3′ end introduced astrong transcriptional terminator (LAT, from the Bacillus licheniformisα-amylase gene) after the end of the BBI gene with a 3′ HindIII site forcloning into the pJM103 vector.

PCR fragments with or without a C-terminal His-Tag were generated withthe primers BBIfusion_FW (SEQ ID NO:14) and BBIHISHindIII_RV (SEQ IDNO:16), or BBIfusion_FW (SEQ ID NO:14) and BBI-HindIII_RV (SEQ IDNO:15), respectively, using the synthetic BBI gene as a template. Unlessindicated otherwise, PCR reactions were typically performed on athermocycler for 30 cycles with High Fidelity Platinum Taq polymerase(Invitrogen) according to the instructions of the supplier (with anannealing temperature of 55° C.). The PCR fragments were cloned asBamHI-HindIII fragments into pJM103 carrying the aprE-BCE103 expressioncassette. The correct gene sequence was verified by DNA sequencing.

Thus, as shown in FIG. 1, the N-terminus of the mature coding region ofthe BBI gene (with or without the His-Tag) was fused in frame to theC-terminal coding region of the first CBD (cellulose binding domain)linker sequence coded by the BCE103 cellulase gene. Thereby, the twoCBD's of BCE103 (Shaw et al., supra) are replaced by BBI in the finalexpression vectors, pJM103BBI or pJM103BBIhis (See, FIG. 2). The aprEpromoter controls the expression of the BCE103-BBI gene fusions (See,Ferrari et al., J. Bact., 170:289-295 [1988]; and Henner et al., J.Bact., 170: 296-300 [1988]).

Competent Bacillus subtilis cells, BG3934comK (degU^(Hy)32, oppA,ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr,amyE::xylRPxylAcomK-phleo), were transformed with the expressionplasmids, pJM103BBI or pJM103BBIhis. The bacteria were made competent bythe induction of the comK gene under control of a xylose induciblepromoter (Hahn et al., Mol. Microbiol., 21:763-775 [1996]). Thetransformants were selected on Luria Broth agar (LA) plates containing 5μg/ml chloramphenicol. To increase the expression by gene amplification,colonies were streaked and grown several times on LA plates with 25μg/ml chloramphenicol until the growth rate with the antibiotic wassimilar to growth rate in the absence of chloramphenicol. The BCE103-BBIfusion protein was produced by growth in shake flasks at 37° C. in TSBmedium (Tryptone Soya Broth from OXOID, 30 g/L) or in MBD medium, a MOPSbased defined medium. MBD medium was made essentially as described(Neidhardt et al., J. Bacteriol., 119: 736-747 [1974]), except NH₄Cl₂,FeSO₄, and CaCl₂ were left out of the base medium, 3 mM K₂HPO₄ was used,and the base medium was supplemented with 60 mM urea, 75 g/L glucose,and 1% soytone. Also, the micronutrients were made up as a 100× stockcontaining in one liter, 400 mg FeSO₄.7H₂O, 100 mg MnSO₄.H₂O, 100 mgZnSO₄.7H₂O, 50 mg CuCl₂.2H₂O, 100 mg CoCl₂.6H₂O, 100 mg NaMoO₄.2H₂O, 100mg Na₂B₄O₇.10H₂O, 10 ml of 1M CaCl₂, and 10 ml of 0.5 M sodium citrate.

BCE103-BBI fusion protein could be easily visualized in samples fromcell free supernatants (after 24 h of growth in TSB medium or 48 h inMBD medium) as the major protein band on SDS-PAGE gels (10% NuPAGE inMES buffer, run as described by the manufacturer, Invitrogen) running at˜44 kDa by using standard protein stains (e.g. GelCode Blue StainReagent; Pierce). The identity of the BCE103-BBI fusion protein wasverified by immunoblots of SDS-PAGE gels using the protocols supplied bythe manufacturer (BM Chromogenic Western Blotting Kit; Roche AppliedScience using an anti-HisTag antibody or an anti-BCE103 cellulasepolyclonal antibody for detection).

To determine the BCE103 activity, cellulase degradation was assessedqualitatively on LA cellulase indicator plates (with 1%carboxymethylcellulose stained with 0.2% Congo Red, or with 0.5%azo-CM-cellulose, Megazyme), or quantitatively by a direct assay inAssay Buffer (100 mM Tris pH 8.6, 0.005% Tween-80) on the culture brothusing a the synthetic substrate, 4-nitrophenyl □-D-cellobioside (Sigma),using methods known in the art (See e.g., van Tilbeurgh et al., Meth.Enzymol., 160:45-59 [1988]).

Trypsin inhibitory assays were performed in Assay Buffer to determinethe BBI activity. Specifically, a standard curve was generated by makingeleven 1:1 serial dilutions (100 μL BBI+100 μL Assay Buffer) of a 2pg/mL standard BBI solution. The BBI standard was purified from a 1mg/ml Trypsin-Chymotrypsin Inhibitor (Sigma Cat. #T-9777) solution in 20mM MES pH 6.0 using a hydrophobic interaction column (POROS HP2, Phenylcolumn, Applied Biosystems). The column was equilibrated with 20 mM MESpH 6.0, loaded with 5 mg of the inhibitor, washed with the equilibrationbuffer, and then the BBI was eluted with water. Unknown BBI samples tobe tested in the inhibitory assay were diluted as necessary, so that twoor more data points would fall within the standard curve (usually 1:10,1:100, 1:200, 1:1000, 1:2000 sample dilutions were tested and then thedilutions fine tuned if necessary). Each diluted BBI standard or sample,20 μL, was added to 80 μL of 50 ng/ml bovine pancreatic trypsin(Worthington) (made by diluting a stock 1 mg/mL trypsin solution intoAssay Buffer). For convenience, the standards and samples were arrayedin 96 well microtiter plates. The reactions were mixed and incubated 15min at 25° C. After the incubation, 100 μL of the 0.5 mg/ml trypsinsubstrate (diluted in Assay Buffer from a 100 mg/ml solution in DMSO),Suc-AAPR-pNA (succinyl-Ala-Ala-Pro-Arg-para-nitroanilide, Bachem), wasadded, mixed and the OD (A₄₀₅) was monitored for 15 min, with 1 timepoint recorded every 12 sec using a Spectra Max 250 (Molecular Devices).The data points were used to determine the Vmax for each reaction. Thestandard curve was generated by plotting Vmax versus BBI concentrationand was fitted to a four-parameter curve. All data fitting was doneusing software supplied by the manufacturer (Molecular Devices). The BBIconcentration of the unknown samples was calculated from the standardcurve. Alternatively, the BBI activity was measured using the sameprotocol but by determining bovine pancreatic chymotrypsin (Worthington)inhibition (chymotrypsin was used at the same concentration as trypsinand chymotrypsin activity was measured by adding 100 μL of a 0.4 mg/mlchymotrypsin substrate, succinyl-Ala-Ala-Pro-Phe-para-nitroanilide,Bachem).

Titers from shake flask runs (500 ml MBD medium in 2.8 L Fernbach 6baffled flasks, 37° C., 225 rpm, harvested 60 h after of growth)typically ranged from 0.4-0.9 mg/ml BCE activity and 40-150 μg/ml BBItrypsin inhibitory activity. However, it is contemplated that titerslikely could be improved further by optimizing the bacterial strain,culture medium and growth conditions (aeration, temperature, time ofharvest, etc.).

In addition to the BCE103 fusion to wild-type BBI, fusion proteins toBBI variants and fusion proteins with various linkers between BCE103 andBBI were produced using the methods outlined above, as described in thefollowing Examples. In addition, fusion proteins were also produced whenthe BBI was fused to the 2^(nd) CBD linker (BCE-cbdD-BBI; See, Example4) making it possible to use the 1^(st) CBD to aid in the purificationprocess.

Example 2 Production of Peptides Substituted into the BBI Reactive SiteLoops as BCE103-BBI Fusion Proteins

In this Example, experiments conducted to produce peptides substitutedinto the BBI reactive site loops as BCE103-BBI fusion proteins aredescribed. The primers, as well as other sequences used in the varioussteps of these experiments are provided below. The sequence of 12BBIck81from the BCE103 fusion site (at the BamHI) to the end of the gene isprovided in FIG. 3. The CK37281 peptide sequences (ACYNLYGWTC (SEQ IDNO:9) are inserted into both the trypsin and chymotrypsin inhibitoryloops.

The primers used to introduce an EcoRI site in the BBI gene usingQuikChange® site-directed mutagenesis (Stratagene) were:

BowBeco-F 5′-GATATGCGTCTGAATTCCTGTCATAGTGCAT (SEQ ID NO: 17) BowBeco-R5′-ATGCACTATGACAGGAATTCAGACGCATATC (SEQ ID NO: 18)

The sequences of the DNA oligonucleotides that were annealed and clonedin the BBI gene (SacI-EcoRI) to replace the trypsin inhibitory loop withthe VegF binding peptide CK37281 were:

1BBck81+ (SEQ ID NO: 19)5′-CTAAACCCTGTTGCGATCAATGCGCATGTTATAATTTGTATGGGTGGACTTGTCGCTGCAGCGATATGCGTCTG 1BBck81− (SEQ ID NO: 20)5′-AATTCAGACGCATATCGCTGCAGCGACAAGTCCACCCATACAAATTATAACATGCGCATTGATCGCAACAGGGTTTAGAGCT

The sequences of the DNA oligonucleotides that were annealed and clonedin the BBI gene (EcoRI-SalI) to replace the chymotrypsin inhibitory loopwith the VegF binding peptide CK37281 were:

2BBck81+ (SEQ ID NO: 21)5′-AATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTA CGGGTGGACCTGTTTTTGCG2BBck81− (SEQ ID NO: 22)5′-TCGACGCAAAAACAGGTCCACCCGTACAGGTTATAACATGCGCAGCT TTTGCAGGCACTATGACAGG

The DNA sequences of the oligonucleotide pairs used to make cassettes tointroduce peptides into the trypsin (SacI and EcoRI restriction sites)or chymotypsin (EcoRI and SalI restriction sites) reactive site loops ofthe synthetic BBI gene are provided below. These peptide codingsequences were then moved into the p2JM103BBI expression vector asSacI-SalI fragments.

Comstatin (1^(st) loop) (SEQ ID NO: 23)CTAAACCCTGTTGCGATCAATGCGCATGTGTTGTTCAGGACTGGGGTCACCACCGTTGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO: 24)AATTCAGACGCATATCGCTGCAGCGACAACGGTGGTGACCCCAGTCCTGAACAACACATGCGCATTGATCGCAACAGGGTTTAGAGCT Comstatin (2^(nd) loop) (SEQ IDNO: 25) CAAAAGCTGTATCTGCGTTGTTCAGGACTGGGGTCACCACCGTTGTTTTT GCG and (SEQID NO: 26) TCGACGCAAAAACAACGGTGGTGACCCCAGTCCTGAACAACGCAGATACAGCTTTTGCATG C2c (1^(st) loop) (SEQ ID NO: 27)CTAAACCCTGTTGCGATCAATGCAGCTGTGGTCGTAAAATCCCGATCCAGTGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO: 28)AATTCAGACGCATATCGCTGCAGCGACACTGGATCGGGATTTTACGACCACAGCTGCATTGATCGCAACAGGGTTTAGAGCT C3c (1^(st) loop) (SEQ ID NO: 29)CTAAACCCTGTTGCGATCAATGCGGTTGTGCTCGTTCTAACCTGGACGAATGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO: 30)AATTCAGACGCATATCGCTGCAGCGACATTCGTCCAGGTTAGAACGAGCACAACCGCATTGATCGCAACAGGGTTTAGAGCT C4c (1^(st) loop) (SEQ ID NO: 31)CTAAACCCTGTTGCGATCAATGCGGTTGTCAGCGTGCTCTGCCGATCCTGTGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO: 32)AATTCAGACGCATATCGCTGCAGCGACACAGGATCGGCAGAGCACGCTGACAACCGCATTGATCGCAACAGGGTTTAGAGCT C5c (1^(st) loop) (SEQ ID NO: 33)CTAAACCCTGTTGCGATCAATGCCAGTGTGGTCGTCTGCACATGAAAACCTGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO: 34)AATTCAGACGCATATCGCTGCAGCGACAGGTTTTCATGTGCAGACGACCACACTGGCATTGATCGCAACAGGGTTTAGAGCT Xa1 (2^(nd) loop) (SEQ ID NO: 35)AATTCCTGTCATAGTGCCTGCAAAAGCTGTATCTGCGCCCGTAGTTTGCC AGCTCAATGTTTTTGCG and(SEQ ID NO: 36) TCGACGCAAAAACATTGAGCTGGCAAACTACGGGCGCAGATACAGCTTTTGCAGGCACTATGACAGG hSCC1 (1^(st) loop) (SEQ ID NO: 37)CTAAACCCTGTTGCGATCAATGCAACTGTACGTACTCAACCCCTCCACAGTGTCGCTGCAGCGATATGCGTCTG and (SEQ ID NO: 38)AATTCAGACGCATATCGCTGCAGCGACACTGTGGAGGGGTTGAGTACGTACAGTTGCATTGATCGCAACAGGGTTTAGAGCT

The DNA sequences of oligonucleotide primer pairs used to introducepeptide sequences into the trypsin or chymotrypsin reactive site loopsusing a QuikChange® II XL site-directed mutagenesis kit (Stratagene) areprovided below. The reactions were performed as outlined by themanufacturer and described in this Example. Twenty cycles were performedwith extensions of 6 minutes at 68° C., denaturations of 50 s at 95° C.,and annealings at 55° C. for 50 s. After the cycles, a final extensionwas performed at 68° C. for 20 minutes.

1A (2^(nd) loop) (SEQ ID NO: 39)CTGTATCTGCAAACGCTCAAAATCTCGTGGCTGTTTTTGCGTCGACATCA C and (SEQ ID NO: 40)CGCAAAAACAGCCACGAGATTTTGAGCGTTTGCAGATACAGCTTTTGCAT G 2B (2^(nd) loop)(SEQ ID NO: 41) CTGTATCTGCTGGTATAATCAAATGACAACATGTTTTTGCGTCGACATCA C and(SEQ ID NO: 42) CGCAAAAACATGTTGTCATTTGATTATACCAGCAGATACAGCTTTTGCAT G 4A(2^(nd) loop) (SEQ ID NO: 43)CTGTATCTGCCATCAACTTGGCCCGAATTCATGTTTTTGCGTCGACATCA C and (SEQ ID NO: 44)CGCAAAAACATGAATTCGGGCCAAGTTGATGGCAGATACAGCTTTTGCAT G 5A (2^(nd) loop)(SEQ ID NO: 45) CTGTATCTGCCATCCGTGGGCACCGTATTCTTGTTTTTGCGTCGACATCA C and(SEQ ID NO: 46) CGCAAAAACAAGAATACGGTGCCCACGGATGGCAGATACAGCTTTTGCAT G6-1A (2^(nd) loop) (SEQ ID NO: 47)CTGTATCTGCAATCTTCATTATCTTCAACAGTGTTTTTGCGTCGACATCA C and (SEQ ID NO: 48)CGCAAAAACACTGTTGAAGATAATGAAGATTGCAGATACAGCTTTTGCAT G 7A (2^(nd) loop)(SEQ ID NO: 49) CTGTATCTGCACACCGTCTCTTTATCGCCCGTGTTTTTGCGTCGACATCA C and(SEQ ID NO: 50) CGCAAAAACACGGGCGATAAAGAGACGGTGTGCAGATACAGCTTTTGCAT G 8B(2^(nd) loop) (SEQ ID NO: 51)CTGTATCTGCCTTACAGATCAATCTAAACCGTGTTTTTGCGTCGACATCA C and (SEQ ID NO: 52)CGCAAAAACACGGTTTAGATTGATCTGTAAGGCAGATACAGCTTTTGCAT G 9A (2^(nd) loop)(SEQ ID NO: 53) CTGTATCTGCGTTACAACATCAATGGGCATGTGTTTTTGCGTCGACATCA C and(SEQ ID NO: 54) CGCAAAAACACATGCCCATTGATGTTGTAACGCAGATACAGCTTTTGCAT G 10B(2^(nd) loop) (SEQ ID NO: 55)CTGTATCTGCCGCGCATCACCGTATGATTGGTGTTTTTGCGTCGACATCA C and (SEQ ID NO: 56)CGCAAAAACACCAATCATACGGTGATGCGCGGCAGATACAGCTTTTGCAT G 11-1A (2^(nd) loop)(SEQ ID NO: 57) CTGTATCTGCTCAACACAAAAAATTCCGCAATGTTTTTGCGTCGACATCA C and(SEQ ID NO: 58) CGCAAAAACATTGCGGAATTTTTTGTGTTGAGCAGATACAGCTTTTGCAT G 12B(2^(nd) loop) (SEQ ID NO: 59)CTGTATCTGCACACAATTTCGCTCTGCAACATGTTTTTGCGTCGACATCA C and (SEQ ID NO: 60)CGCAAAAACATGTTGCAGAGCGAAATTGTGTGCAGATACAGCTTTTGCAT G 13A (2^(nd) loop)(SEQ ID NO: 61) CTGTATCTGCCCGGATCATGTTCCGCATCTTTGTTTTTGCGTCGACATCA C and(SEQ ID NO: 62) CGCAAAAACAAAGATGCGGAACATGATCCGGGCAGATACAGCTTTTGCAT G15-1A (2^(nd) loop) (SEQ ID NO: 63)CTGTATCTGCTCAGGCTTTCCGCTTTCTACATGTTTTTGCGTCGACATCA C and (SEQ ID NO: 64)CGCAAAAACATGTAGAAAGCGGAAAGCCTGAGCAGATACAGCTTTTGCAT G 1A6 (1^(st) loop)(SEQ ID NO: 65) TCAATGCGCATGTGAAGAGATCTGGACTATGCTTTGCCGGTGTTCCGATATGCGTC and (SEQ ID NO: 66)CGGAACACCGGCAAAGCATAGTCCAGATCTCTTCACATGCGCATTGATCG CAACAG 1A6 (2^(nd)loop) (SEQ ID NO: 67) CAAAAGCTGTGCTTGTGAAGAGATCTGGACTATGCTTTGCTTTTGCGTCGACATCACGG and (SEQ ID NO: 68)ACGCAAAAGCAAAGCATAGTCCAGATCTCTTCACAAGCACAGCTTTTGCA TGCACTATG 1C2 (1^(st)loop) (SEQ ID NO: 69) TCAATGCGCATGTTGGGCCCTTACTGTCAAAACATGCCGGTGTTCCGATATGCGTC and (SEQ ID NO: 70)CGGAACACCGGCATGTTTTGACAGTAAGGGCCCAACATGCGCATTGATCG CAACAGG 1C2 (2^(nd)loop) (SEQ ID NO: 71) CAAAAGCTGTGCTTGTTGGGCCCTTACTGTCAAAACATGCTTTTGCGTCGACATCACGG and (SEQ ID NO: 72)ACGCAAAAGCATGTTTTGACAGTAAGGGCCCAACAAGCACAGCTTTTGCA TGCACTATG 2E2 (1^(st)loop) (SEQ ID NO: 73) TCAATGCGCATGTCTTACAGTACTGTGGACTACATGCCGGTGTTCCGATATGCGTC and (SEQ ID NO: 74)CGGAACACCGGCATGTAGTCCACAGTACTGTAAGACATGCGCATTGATCG CAACAGG 2E2 (2^(nd)loop) (SEQ ID NO: 75) CAAAAGCTGTGCTTGTCTTACAGTACTGTGGACTACATGCTTTTGCGTCGACATCACGG and (SEQ ID NO: 76)ACGCAAAAGCATGTAGTCCACAGTACTGTAAGACAAGCACAGCTTTTGCA TGCACTATG 2E5 (1^(st)loop) (SEQ ID NO: 77) TCAATGCGCATGTACTCTTTGGAACAGATCTCCTTGCCGGTGTTCCGATATGCGTC and (SEQ ID NO: 78)CGGAACACCGGCAAGGAGATCTGTTCCAAAGAGTACATGCGCATTGATCG CAACAGG 2E5 (2^(nd)loop) (SEQ ID NO: 79) CAAAAGCTGTGCTTGTACTCTTTGGAATCGATCTCCTTGCTTTTGCGTCGACATCACGG and (SEQ ID NO: 80)ACGCAAAAGCAAGGAGATCGATTCCAAAGAGTACAAGCACAGCTTTTGCA TGCACTATG FGFns(1^(st) loop) (SEQ ID NO: 81)TCAATGCGCATGTACAAACATCGATTCTACTCCTTGCCGGTGTTCCGATA TGCGTC and (SEQ IDNO: 82) CGGAACACCGGCAAGGAGTAGAATCGATGTTTGTACATGCGCATTGATCG CAACAGG FGFns(2^(nd) loop) (SEQ ID NO: 83)CAAAAGCTGTGCTTGCACAAACATCGATTCTACTCCTTGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 84) ACGCAAAAACAAGGAGTAGAATCGATGTTTGTGCAAGCACAGCTTTTGCA TGCACTATGFGFkr (1^(st) loop) (SEQ ID NO: 85)TCAATGCGCATGTACAAAAATCGATCGTACTCCTTGCCGGTGTTCCGATA TGCGTC and (SEQ IDNO: 86) CGGAACACCGGCAAGGAGTACGATCGATTTTTGTACATGCGCATTGATCG CAACAGG FGFkr(2^(nd) loop) (SEQ ID NO: 87)CAAAAGCTGTGCTTGCACAAAAATCGATCGTACTCCTTGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 88) ACGCAAAAACAAGGAGTACGATCGATTTTTGTGCAAGCACAGCTTTTGCA TGCACTATGFGFhI (1^(st) loop) (SEQ ID NO: 89)TCAATGCGCATGTCACCTGCAGACAACTGAAACATGCCGGTGTTCCGATA TGCGTC and (SEQ IDNO: 90) CGGAACACCGGCATGTTTCAGTTGTCTGCAGGTGACATGCGCATTGATCG CAACAGG FGFhI(2^(nd) loop) (SEQ ID NO: 91)CAAAAGCTGTGCTTGCCACCTGCAGACAACTGAAACATGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 92) ACGCAAAAACATGTTTCAGTTGTCTGCAGGTGGCAAGCACAGCTTTTGCA TGCACTATGFGFgy (1^(st) loop) (SEQ ID NO: 93)TCAATGCGCATGTGGCTACTTCATCCCATCGATTTGCCGGTGTTCCGATA TGCGTC and (SEQ IDNO: 94) CGGAACACCGGCAAATCGATGGGATGAAGTAGCCACATGCGCATTGATCG CAACAGG FGFgy(2^(nd) loop) (SEQ ID NO: 95)CAAAAGCTGTGCTTGCGGCTACTTCATCCCATCGATTTGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 96) ACGCAAAAACAAATCGATGGGATGAAGTAGCCGCAAGCACAGCTTTTGCA TGCACTATGMM005 (1^(st) loop) (SEQ ID NO: 97)TCAATGCGCATGTTTACGTATCCTTGCTAACAAATGCCGGTGTTCCGATA TGCGTC and (SEQ IDNO: 98) CGGAACACCGGCATTTGTTAGCAAGGATACGTAAACATGCGCATTGATCG CAACAGG MM005(2^(nd) loop) (SEQ ID NO: 99)CAAAAGCTGTGCTTGCTTACGTATCCTTGCTAACAAATGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 100) ACGCAAAAACATTTGTTAGCAAGGATACGTAAGCAAGCACAGCTTTTGCA TGCACTATGMM007 (1^(st) loop) (SEQ ID NO: 101)GCGATCAATGCGCCTGCAGAACTCAACCATATCCTTTATGTCGGTGTTCC GATATGCGTC and (SEQID NO: 102) GGAACACCGACATAAAGGATATGGTTGAGTTCTGCAGGCGCATTGATCGCAACAGGGTTT MM007 (2^(nd) loop) (SEQ ID NO: 103)CAAAAGCTGTGCCTGCAGAACACAACCTTACCCACTTTGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 104) ACGCAAAAACAAAGTGGGTAAGGTTGTGTTCTGCAGGCACAGCTTTTGCA TGCACTATGMM009 (2^(nd) loop) (SEQ ID NO: 105)CAAAAGCTGTGCCTGCCTGTTAACACCTACTCTTAACTGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 106) ACGCAAAAACAGTTAAGAGTAGGTGTTAACAGGCAGGCACAGCTTTTGCA TGCACTATGMM010 (1^(st) loop) (SEQ ID NO: 107)TCAATGCGCATGCGCTCTTCCAACTCATTCTAACTGTCGGTGTTCCGATA TGCGTCT and (SEQ IDNO: 108) CGGAACACCGACAGTTAGAATGAGTTGGAAGAGCGCATGCGCATTGATCG CAACAGGMM010 (2^(nd) loop) (SEQ ID NO: 109)CAAAAGCTGTGCCTGCGCGCTTCCTACACACTCTAACTGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 110) ACGCAAAAACAGTTAGAGTGTGTAGGAAGCGCGCAGGCACAGCTTTTGCA TGCACTATGMM017 (2^(nd) loop) (SEQ ID NO: 111)CAAAAGCTGTGCCTGCCCTTTAGGCCTTTGCCCACCTTGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 112) ACGCAAAAACAAGGTGGGCAAAGGCCTAAAGGGCAGGCACAGCTTTTGCA TGCACTATGFGFps1 (2^(nd) loop) (SEQ ID NO: 113)AAGCTGTATCTGCTGGAACATCGATTCTACACCTTGTTTTTGCGTCGACA TCACGG and (SEQ IDNO: 114) ACGCAAAAACAAGGTGTAGAATCGATGTTCCAGCAGATACAGCTTTTGCA TGCACTFGFps2 (1^(st) loop) (SEQ ID NO: 115)GCGATCAATGCATCTGTACTTGGATTGACAGTACTCCTTGTCGGTGTTCC GATATGCGTC and (SEQID NO: 116) GGAACACCGACAAGGAGTACTGTCAATCCAAGTACAGATGCATTGATCGCAACAGGGTTT FGFps2 (2^(nd) loop) (SEQ ID NO: 117)AAGCTGTATCTGCACATGGATCGATAGTACTCCTTGTTTTTGCGTCGACA TCACGG and (SEQ IDNO: 118) ACGCAAAAACAAGGTGTAGAATCGATCCATGTGCAGATACAGCTTTTGCA TGCACT andFGFpsB (2^(nd) loop) (SEQ ID NO: 119)AAGCTGTATCTGTACATGGATCGATTGGACACCTTGTTTTTGCGTCGACA TCACGG and (SEQ IDNO: 120) ACGCAAAAACAAGGTGTCCAATCGATCCATGTACAGATACAGCTTTTGCA TGCACT 1A8(2^(nd) loop) (SEQ ID NO: 121)CAAAAGCTGCGCATGTGTTACTACAGATTGGATCGAATGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 122) ACGCAAAAACATTCGATCCAATCTGTAGTAACACATGCGCAGCTTTTGCA TGCACTATG1A12 (2^(nd) loop) (SEQ ID NO: 123)CAAAAGCTGTGCCTGCCCAACACTTTGGACTCATATGTGTTTTTGCGTCG ACATCACGGAC and (SEQID NO: 124) ACGCAAAAACACATATGAGTCCAAAGTGTTGGGCAGGCACAGCTTTTGCATGCACTATGAC 1E11 (2^(nd) loop) (SEQ ID NO: 125)CAAAAGCTGCGCATGTTACTACTCTCAATTCCACCAATGTTTTTGCGTCG ACATCACGG and (SEQ IDNO: 126) ACGCAAAAACATTGGTGGAATTGAGAGTAGTAACATGCGCAGCTTTTGCA TGCACTATGTGFps1 (2^(nd) loop) (SEQ ID NO: 127)CAAAAGCTGTCTTTGTCCGGAAAACGATAACGTTTCTCCTTGTAATTGCG TCGACATCACGGACTTCTGand (SEQ ID NO: 128) TGTCGACGCAATTACAAGGAGAAACGTTATCGTTTTCCGGACAAAGACAGCTTTTGCATGCACTATGAC

The DNA sequences of the oligonucleotide pair used to make the cassetteto introduce the MM021 peptide into the chymotrypsin reactive site loopsof the p2JM103-Ink2-BBI expression vector are provided below. Thecassette was ligated into the SphI and SalI restriction sites in thevector.

MM021 (2^(nd) loop) (SEQ ID NO: 129)CAAAAGCTGTGCTTGTAAACACAACGTACGTCTTTTATGTTTTTGCG and (SEQ ID NO: 130)TCGACGCAAAAACATAAAAGACGTACGTTGTGTTTACAAGCACAGCTTTT GCATG

Libraries made of cysteine constrained peptides are popular reagents(e.g. the commercially available PhD-C7C Phage Display Peptide LibraryKit; NEB) for selecting peptides that bind to substrates of interest.BBI has two cysteine constrained reactive site loops that arestructurally similar to the peptide loops displayed in various methodsused to select peptide binders. So, once a cysteine constrained bindingpeptide has been selected, BBI is suitable for use as a scaffold topresent the peptide in a binding reaction.

The VEGF binding peptide CK37281 (See e.g., co-pending U.S. ProvisionalPatent Application Ser. No. 60/520,403, filed Nov. 13, 2003,incorporated herein by reference) was grafted into BBI by replacing thetrypsin, chymotrypsin, or both reactive site loops, with the CK37281peptide sequence (ACYNLYGWTC) (SEQ ID NO:9) by using DNA oligonucleotidecassettes. To facilitate the construction, an EcoRI site was introducedin the coding region of the BBI gene (custom synthesized by OperonTechnologies; See, Example 1) between the trypsin and chymotrypsinreactive site loops by QuikChange® site-directed mutagenesis, usingmethods described by the manufacturer (Stratagene) using the primersBowBeco-F and BowBeco-R, shown above (0.5 pmol of each primer was usedin the QuikChange® reaction; after an initial denaturation step of 97°C. for 3 minutes, 18 PCR cycles of 68° C. for 12 minutes, 95° C. for 30seconds and 55° C. for one minute, followed by a final extensionreaction for 15 minutes at 68° C.).

To replace the trypsin inhibitory peptide loop, two DNA oligonucleotides(IBBCK81+ and IBBCk81−) were annealed and ligated into the SacI andEcoRI restriction sites. Likewise, to replace the chymotrypsininhibitory peptide loop, EcoRI and Sa/I sites were used for insertion ofa DNA cassette made by annealing the oligonucleotides (2BBck81+ and2BBck81−). The CK37281 peptide was grafted into both loops by insertingthe CK37281 peptide in the chymotrypsin loop (using the oligonucleotides(2BBck81+ and 2BBck81−) after the trypsin loop was first replaced by theCK37281 peptide. BBI with the CK37281 peptide in the trypsin loop(1BBIck81) was moved into the pJM103BBI expression vector as a SacI-SphIfragment. BBI with the CK37281 in the chymotrypsin loop (2BBIck81), orboth loops (12BBIck81), was moved into pJM103BBI as SacI-SalI fragments.The correct sequences were verified by DNA sequencing (the sequence of12BBIck81 gene is shown in FIG. 3). The resulting vectors, pJM103-1BBIck81, pJM103-2BBIck81, or pJM103-12BBIck81, were used to transform B.subtilis BG3934comK, and the production of the BCE fusion proteins wasdetermined as in Example 1 above.

The fusion protein running at ˜44 kDa was detected by SDS-PAGE to be themajor protein present in the cell free broth. Although in some cases,there was significant degradation (up to 50%) of the BBI moiety(especially after >48 h of growth in MBD medium), as observed by thepresence of a prominent protein band running at ˜34 kDa corresponding tothe BCE103 catalytic core. In these cases, the titers of the BCE103cellulase were similar to that measured with fusions to the wild-typeBBI (Example 1), but the activity of the BBI (trypsin inhibition with2BBIck81, or chymotrypsin inhibition with 1BBIck81) was generally abouttwo fold less.

To reduce the proteolytic degradation of BBI variants during growth(i.e. decrease the amount of BCE103 cellulase core present on SDS-PAGEgels in comparison to the fusion protein), a Bacillus subtilis strainwith nine protease genes deleted, BG6006 (degU^(Hy)32, oppA,ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr ΔwprA, Δmpr-ybjF,ΔnprB, amyE::xylRPxylAcomK-ermC), was used as an expression host, andthe growth temperature (35° C.) and aeration (200 rpm) were reduced.With these changes, a major fusion protein band (˜44 kDa) was observedon SDS-PAGE gels with an insignificant band present at the molecularweight expected for the BCE catalytic core protein (˜34 kDa).

In addition to the CK37281 peptide, a number of other cysteineconstrained peptides were produced when substituted into the trypsinand/or chymotrypsin reactive site loops of BBI fused to the C-terminusof the BCE103 cellulase. Specific examples included:

Peptides designed or selected as complement antagonists, compstatinintroduced into the 1^(st) or 2^(nd) reactive site loops (See, Sahu etal., J. Immunol., 157: 884-891, [1996]), C2c (1^(st) loop), C3c (1^(st)loop), C4c (1St loop) and C5c (1^(st) loop); or peptides selected in aFactor B binding reaction 1B, 2B, 4A, 5A, 6-1A, 7A, 8B, 9A, 10B, 11-1A,12B, 13A, and 15-1A (all in 2^(nd) loop);

Peptides designed to bind to the proteases Factor Xa or stratum corenumchymotrypsin, Xa1 (2^(nd) loop) or hSCC1 (1^(st) loop), respectively;

Peptides selected in FGF5 binding reactions 1A6 (1^(st) or 2nd loop),1C2 (1^(st) or 2nd loop), 2E2 (1^(st) or 2nd loop), 2E5 (1^(st), 2^(nd)or both loops), FGFns (1^(st) or 2^(nd) loop), FGFkr (1^(st) or 2^(nd)loop), FGFhl (1^(st) or 2^(nd) loop), FGFgy (1^(st) or 2^(nd) loop),MM005 (1^(st) or 2^(nd) loop), MM007 (1^(st), 2^(nd) or both loops),MM009 (2^(nd) loop), MM010 (1^(st), 2^(nd) or both loops), MM017 (2^(nd)loop), FGFps1 (2^(nd) loop), FGFps2 (1^(st), 2^(nd) or both loops), andFGFpsB (2^(nd) loop); and

Peptides selected in TGFβ-L binding reactions 1A8 (2^(nd) loop), 1A12(2^(nd) loop), 1E11 (2^(nd) loop), TGFps1 (2^(nd) loop), and MM021(2^(nd) loop).

The oligonucleotides used to introduce these peptides into either thetrypsin (1^(st) loop) or chymotrypsin (2^(nd) loop) reactive site loops,and methods used to graft these peptides into BBI, are provided above.In all cases, fusion proteins were produced as determined by SDS-PAGEgels. However, with some substituted peptides, the amount of intactfusion protein was increased by reducing the proteolytic degradation asdescribed above for the CK37281 substituted peptide.

Example 3 Activation of BBI by Thiol Reducing/Oxidizing Agents

After growth, the activity of the BBI (by trypsin or chymotrypsininhibition) is typically some 5-20 times lower than what would beexpected from the activity of the BCE103 cellulase measured in the cellfree supernatants (the two molecules should be present at a 1:1 molarratio in the fusion protein). An increase in the activity of BBI(measured by either trypsin or chymotrypsin inhibition) in theBCE103-BBI fusion protein can be routinely obtained by adding □ME,typically concentrations of 1-4 mM added to the MBD growth medium about14 h after inoculation. The trypsin or chymotrypsin inhibitory activityof BBI in the fusion protein is also lower than expected when bindingpeptides (e.g. VegF binding peptide CK37281) replace the chymotrypsin ortrypsin reactive site loop, respectively. As with the wild-type BBI, theinhibitory activity can be increased by treatment with bME.Unexpectedly, other thiol reducing agents (e.g., cysteine, reducedglutathione, DL-dithiothreitol and Tris[2-carboxyethyl]phosphine) hadsmall or negligible effects on the activation of BBI during growth inthese experiments. Also, additions of antioxidants (e.g., ascorbic acidor DL-α-tocopherol acetate) or other adjuvants to the growth medium(e.g., isoleucine, soybean oil, Tween-80), or growth at 30° C. did notsignificantly improve the BCE103:BBI activity ratio.

Specifically, to determine the BBI activation during growth, cultures ofB. subtilis BG6006 transformed with p2JM103-E3-2BBIck81 (See, Example 4,below) were grown in 40 ml MBD medium in 250 ml shake flasks at 37° C.for 13 h. Then, the thiol reducing agents indicated on the graph in FIG.4 were added and cell supernatants harvested after 62 h of growth. Thereagents 2-mercaptoethanol (BME), cysteine (Cys), reduced glutathione(Glut), and DL-dithiothreitol (DTT) were added to the growth medium tothe final concentrations indicated on the graph provided in FIG. 4.Concentrations of 5 mM BME can result in better BCE103:BBI activityratios but typically result in an overall decrease in both BCE103 andBBI titers (see FIG. 4), at least partially due to the reduction inbacterial growth caused by the added reagent. Titers of BCE103 and2BBIck81 were determined using the assays described in Example 1.

BBI activation was also achieved after partial purification of thefusion proteins (e.g. BCE-Ink2-2BBIck81, see Example 4 below) byQ-Sepharose ion exchange chromatography.

The fusion protein was purified from cell free broth obtained from shakeflasks or fermentor runs. The broth was filtered, diluted five to tenfold in water and the pH adjusted to pH 7.5-8.0. The diluted sample wasloaded onto a column packed with Q-Sepharose resin (GE Healthcare). Thecolumn was washed with 50 mM Tris pH 7.5 and then washed again in thesame buffer containing 300 mM NaCl. The fusion protein was eluted in thesame buffer with 700 mM NaCl.

To activate the BBI, the pooled fusion protein fractions were dilutedten fold in Assay Buffer then treated with 2 mM BME and 0.2 mM oxidizedglutathione (GSSG) with constant mixing on a stir plate or rockerplatform for about 24 h at room temperature. The BBI could generally beactivated to about 70-100% of the expected trypsin inhibitory activitybased on the measured concentration of the BCE103 cellulase. Althoughthe activation method outlined above generally yielded the best results,in some cases, in order to maximize the activation of a given sample,screens were performed in 96-well plates to determine the optimalconditions. Initially, the typical conditions screened were the dilutionin Assay Buffer (e.g., a 2-50 fold dilution series), BME concentration(e.g., series between 0.5-5 mM) and oxidized glutathione concentration(e.g. 0 mM then a series of 1/20 to ½ the BME concentration).

The activation of the fusion protein BCE-Ink2-2BBIck81 is shown in FIG.5. In this specific example, the fusion protein from a Q-Sepharosepurification was diluted 1:10 in Dulbecco's PBS (Mediatech) with 0.005%TWEEN®-80. Beta-mercaptoethanol was added to a final concentration of 3mM and incubated overnight at room temperature on a rocker. The samplewas further incubated at room temperature for about 60 h with vigorousstirring on a magnetic stir plate. The titers of the BCE103 and 2BBIck81(before and after βME treatment) were determined by cellulase assays andtrypsin inhibitory assays, respectively.

In some embodiments, such as for activating BBI or it variants in cellfree broth from large volume fermentations, it is desirable to reducethe dilution and βME concentration in the activation reaction. This canbe accomplished by using higher concentrations of buffer (500 mM Tris pH8.6), or changing to zwitterionic buffers such as CHES (also CAPS,Tricine, TAPS, and other suitable zwitterionic buffers). For example,cell free broth (or fusion protein fractions purified by ion exchangechromatography) was diluted 1:1 in 375 mM CHES pH 8.6 with 0.005%TWEEN®-80 then activated with 1 mM BME and 10 mM Na₂SO₃ and incubatedwith stirring at room temperature for about 24 h. BBI or its variants,as BCE103 cellulase fusion proteins, were routinely activated by thismethod to 70-100% of the expected value (based on BCE103 cellulaseactivities).

Example 4 Release of Free BBI/Variants by Cleavage of the BCE103-BBIFusion Proteins

This Example describes experiments developed to release free BBI or itsvariants by cleavage of the BCE103-BBI fusion proteins.

The sequences of the DNA oligonucleotide pairs that were annealed andligated into the BamHI and SacI sites of pJM103-BBI to generatepotential cleavage sites during culture growth between the BCE103catalytic domain and BBI are provided below.

BCEsubBBI (a subtilisin-type sensitive peptide sequence) (SEQ ID NO:131) GATCCAGGTGGAGCTGCTTTAGTTGACGATGAGAGCT and (SEQ ID NO: 132)CTCATCGTCAACTAAAGCAGCTCCACCTG BCEcbdLBBI (a portion of the 1^(st) CBD)(SEQ ID NO: 133) GATCCAGGTGAACCTGACCCAACTCCTCCATCTGATCCTGGAGAATACCCAGCTTGGGACGATGAGAGCT and (SEQ ID NO: 134)CTCATCGTCCCAAGCTGGGTATTCTCCAGGATCAGATGGAGGAGTTGGGT CAGGTTCACCTGBCEproBBI (the entire pro peptide of BBI) (SEQ ID NO: 135)GATCCGGCGAACCTGCGTCTGTCTAAGCTTGGCCTGCTTATGAAATCAGACCATCAGCACAGCAATGACGATGAGAGCT and (SEQ ID NO: 136)CTCATCGTCATTGCTGTGCTGATGGTCTGATTTCATAAGCAGGCCAAGCT TAGACAGACGCAGGTTCGCCGBCEshortproBBI (a C-terminal portion of the pro peptide of BBI) (SEQ IDNO: 137) GATCCAAAATCAGACCATCAGCACAGCAATGACGATGAGAGCT and (SEQ ID NO:138) CTCATCGTCATTGCTGTGCTGATGGTCTGATTTTG

The sequences of the DNA oligonucleotide pair that was annealed andligated into the BamHI and SacI sites of p2JM103-BBI to fuse BBI to the2^(nd) CBD linker of BCE103 cellulase are provided below.

BCEcbdDBBI (SEQ ID NO: 139)GATCCAGGAGAACCGGACCCAACGCCCCCAAGTGATCCAGGAGAGTATCCAGCATGGGATTCAAATCAAATTTACACAAATGAAATTGTGTATCATAACGGTCAGTTATGGCAAGCGAAATGGTGGACACAAAATCAAGAGCCAGGTGACCCATACGGTCCGTGGGAACCACTCAAATCTGACCCAGATTCAGACGATGA GAGCT and (SEQ ID NO:140) CTCATCGTCTGAATCTGGGTCAGATTTGAGTGGTTCCCACGGACCGTATGGGTCACCTGGCTCTTGATTTTGTGTCCACCATTTCGCTTGCCATAACTGACCGTTATGATACACAATTTCATTTGTGTAAATTTGATTTGAATCCCATGCTGGATACTCTCCTGGATCACTTGGGGGCGTTGGGTCCGGTTCTCCTG

The peptide sequences susceptible to acid cleavage between aspartic acidand proline residues are provided below.

Linker 1 - WGDPHY (SEQ ID NO: 141) (Lidell et al., J. Biol. Chem. 278:13944-51 [2003]) Linker 2 - DNNDPI (SEQ ID NO: 142) (Segalas et al.,FEBS Lett., 371: 171-175 [1995]) Linker 3 - VVADPN (SEQ ID NO: 143(Kemperman et al., Appl. Env. Microbiol., 69: 1589-1597 [2003])

Oligonucleotide primers used to introduce a BssHII site into pJM103BBIby QuikChange® site-directed mutagenesis are provided below.

BCEbss-F (SEQ ID NO: 144) 5′-TGGCGTTCAGCAACATGAGCGCGCAGGCTGATGATTABCEbss-R (SEQ ID NO: 145) 5′-TAATCATCAGCCTGCGCGCTCATGTTGCTGAACGCCA

Sequences of the DNA oligonucleotides that were annealed as a cassette(SalI-HindIII) to introduce HindIII and XhoI sites after the stop codonof BBI, to introduce a PacI site after the LAT, and remove the originalHindIII site are provided below.

BCEterm+ (SEQ ID NO: 146)5′-GACATCACGGACTTCTGCTATGAGCCATGTAAACCAAGCGAGGACGATAAAGAGAACTAAAAGCTTAACTCGAGGTTAACAGAGGACGGATTTCCTGAAGGAAATCCGTTTTTTTATTTTTAATTAAG BCterm− (SEQ ID NO: 147)5′-AGCTCTTAATTAAAAATAAAAAAACGGATTTCCTTCAGGAAATCCGTCCTCTGTTAACCTCGAGTTAAGCTTTTAGTTCTCTTTATCGTCCTCGCTTGGTTTACATGGCTCATAGCAGAAGTCCGTGATG

PCR primers used to generate the acid labile linkers provided above(i.e., Linker 1, Linker 2, and Linker 3) inserted between the BCE103catalytic domain and BBI are provided below.

BCE103coreBssHII_FW (SEQ ID NO: 148) 5′-CAGCAACATGAGCGCGCAGGCTGlinkerWGDPHY_RV (SEQ ID NO: 149)5′-ATCGTCTGGATCCGGATAGTGGGGGTCTCCCCAAGATGCTGATTCTC TTATTTTTTCClinkerDNNDPI_RV (SEQ ID NO: 150)5′-ATCGTCTGGATCCGGTATGGGATCATTGTTGTCAGATGCTGATTCTC TTATTTTTTCCClinkerVVADPN_RV (SEQ ID NO: 151)5′-ATCGTCTGGATCCGGGTTGGGATCTGCAACTACAGATGCTGATTCTC TTATTTTTTCCC

PCR primers used to generate the acid labile linkers provided above(i.e., Linker 1, Linker 2, and Linker 3) inserted into the 1^(st) CBDlinker.

BCE103corePstI_FW (SEQ ID NO: 152) GCATAAGGAT GAGTCATCTG CAGCGLplusWGDPHY_RV (SEQ ID NO: 153)5′-ATCGTCTGGATCCGGATAGTGGGGGTCTCCCCACGGTTCTCCTGGAT CAGATGGCGGLplusDNNDPI_RV (SEQ ID NO: 154)5′-ATCGTCTGGATCCGGTATGGGATCATTGTTGTCCGGTTCTCCTGGAT CAGATGGCGGLplusVVADPN_RV (SEQ ID NO: 155)5′-ATCGTCTGGATCCGGGTTGGGATCTGCAACTACCGGTTCTCCTGGAT CAGATGGCGG

Protein sequence of the acid labile linkers inserted between the BCE103catalytic domain and BBI are provided below. The acid labile linkers areshown in bold type and the sequences from the first CBD domain areunderlined.

Linker 1 BCE-WGDPHY- PDP-BBI (SEQ ID NO: 156) Linker 2 BCE-DNNDPI-PDP-BBI (SEQ ID NO: 157) Linker 3 BCE-VVADPN- PDP-BBI (SEQ ID NO: 158)LinkerPlus 1 BCE-IPPSDPTPPSDPGEP-WGDPHY- PDP-BBI (SEQ ID NO: 159)LinkerPlus 2 BCE-IPPSDPTPPSDPGEP-DNNDPI- PDP-BBI (SEQ ID NO: 160)LinkerPlus 3 BCE-IPPSDPTPPSDPGEP-VVADPN- PDP-BBI (SEQ ID NO: 161)

The sequences of the DNA oligonucleotide pairs that were annealed andligated into the BamHI and SacI sites of pJM103-BBI to generatepotential cleavage sites between the BCE103 catalytic domain and BBIduring the purification process are provided below.

BCEentBBI (Enteropeptidase cleavage site) (SEQ ID NO: 162)GATCCAGGTGGAGACGACGATGACAAAGACGATGAGAGCT and (SEQ ID NO: 163)CTCATCGTCTTTGTCATCGTCGTCTCCACCTG BCEgenen1BBI (Genenase I cleavage site)(SEQ ID NO: 164) GATCCAGGTGCTGCTCATTACGACGATGAGAGCT and (SEQ ID NO: 165)CTCATCGTCGTAATGAGCAGCACCTG

The sequences of the DNA oligonucleotide pairs that were annealed andligated into the BamHI and SacI sites of pJM103-Ink2-1 BBIck81 togenerate potential cleavage sites between the BCE103 catalytic domainand BBI during the purification process are provided below.

BCEfurinBBI (Furin/Blisterase cleavage site) (SEQ ID NO: 166)GATCCACGTGCTAAAAGAGACGATGAGAGCT and (SEQ ID NO: 167)CTCATCGTCTCTTTTAGCACGTG BCEgenen2BBI (Genenase I cleavage site) (SEQ IDNO: 168) GATCCAGGCGCTGCACACTACAACGACGATGAGAGCT and (SEQ ID NO: 169)CTCATCGTCGTTGTAGTGTGCAGCGCCTG BCEfleBBI (Mpr cleavage site) (SEQ ID NO:170) GATCCATTCCTTGAAGACGATGAGAGCT and (SEQ ID NO: 171)CTCATCGTCTTCAAGGAATG

Sequences of the oligonucleotide primer pairs used to introduce the Eand E3 linkers in Linker 2 by QuikChange site-directed mutagenensis(Stratagene) are provided below.

BCE-EInk-BBI (Mpr cleavage site) CCCATACCGGAGCCAGACGATGAGAGCTC (SEQ IDNO: 172) and CATCGTCTGGCTCCGGTATGGGATCATTGTTG (SEQ ID NO: 173)

The protein sequence of the E3 linker between the BCE103 catalyticdomain and BBI was DNNDPIPEPDDESFNMPIPEP (SEQ ID NO:174). In thissequence, the E Linker is underlined and the sequence generated byfaulty recombination in E. coli is shown in bold type. Cleavage by Mpr(or V8 protease) can occur after any of the three glutamic acids presentin the E3 Linker. Thus, the structure was BCE-(SEQ ID NO:174)-BBI

The sequences of the DNA oligonucleotide pairs that were annealed andligated into the BamHI and SacI sites of p2JM103-Ink2-2BBIck81 togenerate potential Genenase I cleavage sites between the BCE103catalytic domain and BBI are provided below.

BCEgenen3BBI (SEQ ID NO: 175)GATCCAGGCGCTGCACACTACAAATCAGACCATCAGCACAGCAATGACGA TGAGAGCT and (SEQ IDNO: 176) CTCATCGTCATTGCTGTGCTGATGGTCTGATTTGTAGTGTGCAGCGCCTG BCEgenen4BBI(SEQ ID NO: 177) GATCCAGGCGCTGCACACTACGTAGAATTTCAAGACGATGAGAGCT and (SEQID NO: 178) CTCATCGTCTTGAAATTCTACGTAGTGTGCAGCGCCTG

The protein sequence of a Genenase I sensitive cleavage site (also acidand Mpr sensitive) inserted between the BCE103 catalytic domain and BBIwas DNNDPIPDPGAAHYVEFQ (SEQ ID NO:179). The Genenase I site (Gen4Linker) is in bold type (cleavage occurs between the tyrosine andvaline) (NEB) and Linker 2 is underlined. Cleavage by Mpr can also occurafter the glutamic acid that follows the valine in the Gen4 linker. Thesequence used herein was BCE-SEQ ID NO:179)-BBI

Cleavage sites in the BCE103-Ink2-2BBIck81 fusion protein are indicatedbelow. The C-terminal seven amino acids of the BCE103 catalytic domain(underlined), linker 2 sequence (bold type), and 2BBIck81 sequences areshown. The acid/heat labile Asp-Pro bonds are indicated with solidheaded arrows and the Mpr sensitive bonds after glutamic acids areindicated with line headed arrows.

In order to isolate free BBI or its variants, the BBI moiety needs to becleaved from the BCE103-BBI fusion protein. In some embodiments, this isaccomplished during growth, by proteases intrinsically produced by B.subtilis. In some alternative embodiments, this cleavage occurs aftergrowth, during the purification process (e.g. by acid/heat orproteolytic cleavage). Linkers potentially susceptible to cleavageduring growth were designed (See, above, sub, cbdL, pro, shortpro, andcbdD) and cloned into the pJM103BBI or p2JM103BBI expression vectors asBamHI-SacI cassettes. The production of fusion protein versus BCE103catalytic domain was analyzed on SDS-PAGE gels as described in Example1.

Little cleavage of the fusion protein was observed for all these linkersexcept with the pro linker, which was nearly completely cleaved so thatvery little intact fusion protein was observed on gels, although therewas a large band corresponding to the BCE103 catalytic core.Unfortunately, this cleavage during growth resulted in negligible BBIactivity measured in cell free supernatants and no BBI band could beidentified on SDS-PAGE gels. Although it is not intended that thepresent invention be limited to any particular mechanism or theory, itis possible that the BBI is particularly sensitive to proteolyticdegradation in its inactive form. Thus, cleavage during the purificationprocess after activation is generally preferred.

In some embodiments, the bonds between aspartic acid and prolineresidues are cleaved by heat treatment at acidic pH as known in the art(See e.g., Landon, Meth. Enzymol., 47:145-149 [1977]). The 1st CBDlinker in the BCE103 cellulase has three Asp-Pro dipeptide sequences(See, FIG. 1) with the potential to be cleaved by acid/heat treatment.However, cleavage by acid/heat treatment at these sites was found to beinefficient. Protein sequences that are especially labile to acid/heathave been described in the literature, three of such sequences areWGDPHY (SEQ ID NO:141), DNNDPI (SEQ ID NO:142), and VVADPN (SEQ IDNO:143) (i.e., Linkers 1, 2 and 3).

Before these acid labile linkers were introduced into the BCE103-BBIexpression vector, pJM103BBI, a BssHII site was introduced byQuikChange® XL (Stratagene) mutagenesis (using the manufacturer'smethods; and described in Example 2 above, except 8 minute extension and1 minute denaturation steps were used) in the aprE signal sequencecoding region using the oligonucleotide primers BCEbss-F and BCEbss-R(provided above). Then, HindIII and XhoI sites were inserted in front ofthe LAT terminator (after the BBI stop codon) and a PacI site was addedafter the terminator (the original HindIII site after the LAT terminatorwas removed) by inserting an oligonucleotide cassette (BCEterm+ andBCEterm−; provided above) into the SalI and the original HindIII sites.This new vector was called “p2JM103BBI.”

The acid labile linker fragments were generated by PCR, using forwardprimer BCE103coreBssHII_FW with each of the reverse primers, linkerWGDPHY_R, linker DNNDPI_RV, or linker VVADPN_RV (the sequences of whichare all provided above) and p2JM103BBI as the template (see Example 1for the PCR protocol). The PCR fragments of 970 bp were digested withBamHI and PstI, the 154 bp fragments encoding the acid linker fragmentswere isolated from an agarose gel after electrophoresis, and ligatedinto the p2JM103 vector digested with BamHI and PstI that had also beenpurified from a gel. The linker sequences in the final expressionvectors, p2JM103Ink1-BBI, p2JM103Ink2-BBI and p2JM103Ink3-BBI, wereverified by DNA sequencing.

Competent B. subtilis strain BG3934comK or BG6006 were transformed withthe plasmids, colonies selected on 5 μg/ml chloramphenicol LA plates andamplified to 25 μg/ml chloramphenicol as described in Example 1.

Similarly, the acid labile linkers were inserted into the first CBDlinker. Specifically, PCR fragments were generated using the forwardprimer BCE103corePstI_FW with the reverse primers LplusWGDPHY_RV,LplusDNNDPI_RV, or LplusVVADPN_RV (See above, for the sequences) withp2JM103BBI as a template. The PCR fragments of about 150 bp weredigested with BamHI and PstI, purified and ligated to the p2JM103BBIvector digested with BamHI and PstI. The correct sequences were verifiedby DNA sequencing and the plasmids p2JM103pIInk1-BBI, p2JM103pIInk2-BBIand p2JM103pIInk3-BBI were used to transform B. subtilis strains asdescribed above.

After growth in MBD medium, the fusion proteins were purified by ionexchange chromatography essentially as described above (See, Example 2).The fusion protein was cleaved by treatment at 55° C. for 16 h in 10%formic acid. The BCE103 catalytic domain precipitated during the acidtreatment and was removed by centrifugation. The free BBI in thesupernatant was dried overnight on a SpeedVac. The sample was suspendedin 50 mM Tris pH 8 before loading on the SDS-PAGE gel. By analysis ofthe protein stained SDS-PAGE gels, it was observed that acid cleavagewas much more efficient in the fusion proteins where Linker 2 wasinserted between the BCE103 catalytic domain and BBI(BCE-DNNDPI-PDP-BBI). This linker was found to be cleaved in a couple ofhours at 75° C. in 20 mM glycine pH 2.

In alternative embodiments, the fusion protein was cleaved by treatmentwith a protease during the purification process. Linkers were designedwith cleavage sites for glutamic acid specific proteases (e.g., Mpr orV8 protease), Furin/blisterase, Genenase I, and Enteropeptidase(Enterokinase). These linkers were introduced as oligonucleotidecassettes (See above, for the sequences) between the BCE103 catalyticcore and BBI in the expression vector using the BamHI and SacI sites(See, FIG. 1). In the coding region of the original expression vector(pJM103BBI), there is a glutamic acid residue in the 1^(st) CBD domainand at the third residue in BBI (See, FIG. 1), which is contemplated tobe susceptible to cleavage by glutamic acid specific proteases such asB. subtilis Mpr (BsMpr) or V8 protease. However, neither BsMpr nor V8protease were found to cleave the BCE-BBI fusion protein veryefficiently at these sites. Thus, it was necessary to design otherlinkers that were susceptible to cleavage by these proteases.

The six acid labile linkers described above were tested for cleavage byBsMpr. These fusion proteins were cleaved by treatment for 16 h with 16μg of BsMpr at room temperature. After cleavage, the BCE103 catalyticdomain was precipitated by the addition of 10% formic acid and removedby centrifugation. The free BBI in the supernatant was dried overnighton a SpeedVac. The sample was suspended in 50 mM Tris pH 8, beforeloading on the SDS-PAGE. Similar to the acid cleavage, theBCE-DNNDPI-PDP-BBI (Linker 2) fusion protein was much more efficientlycleaved by BsMpr than any of the other linkers. Therefore, BBI and itsvariants were found to be effectively released from theBCE-DNNDPI-PDP-BBI fusion protein either by acid/heat treatment orproteolytic digestion with a glutamic acid specific protease such asBsMpr. Several other linkers designed for cleaved by Mpr (e.g., E, E3linker, and fle, provided above) were tested but none of them had anyadvantages over Linker 2 (the E3 linker was generated by faultyrecombination in E. coli after transformation with the QuikChange®site-directed mutagenesis reaction designed to construct the E linker).As shown above, there are two acid/heat labile cleavage sites in Linker2 and three sites sensitive to cleavage by Mpr.

Linkers designed for cleavage by Furin or Blisterase (NEB)(BCEfurinBBI), or Enteropeptidase (Enterokinase, NEB) (BCEentBBI) weretested, but none of these sequences were cleaved efficiently by theappropriate protease. Four linkers were also designed (BCEgenen1BBI,BCEgenen2BBI, BCEgenen3BBI, and BCEgenen4BBI) and tested for cleavage byGenenase I (NEB). Efficient cleavage of the fusion protein was observedonly with the Gen4 Linker (BCEgenen4BBI). BsMpr was also found toefficiently cleave the Gen4 linker.

After activation of the purified BCE-Ink2-2BBIck81 fusion protein,cleavage by BsMpr does not go to completion as judged by SDS-PAGE gels.However, it was discovered that complete cleavage after activation ofBCE-BBI fusion proteins with Linker 2 (or the Gen4 linker) can beaccomplished by using the Mpr protease isolated from Bacilluslicheniformis (BIMpr). While it is not intended that the presentinvention be limited to any particular mechanism, cleavage after thethird amino acid in mature BBI appeared to be more sensitive to BIMprwhile cleavage after the sixth amino acid from the C-terminus of BBI ismore sensitive to BsMpr cleavage.

In some embodiments, after cleavage, the BBI is purified away from theBCE103 catalytic domain by selective acid precipitation (pH 3 or lower)of the BCE103 catalytic domain as described above, ion exchangechromatography (See, Example 5), or by selective binding of BBI on ananhydrotrypsin-agarose (Sigma) column loaded in 50 mM Tris pH 8.0,washed with 50 mM Tris pH 8.0 with 150 mM NaCl, then eluting bound BBIwith 50 mM glycine pH 2.2 with 300 mM NaCl).

Example 5 Binding of BBIck81 to VegF

In this Example, experiments conducted to assess the binding of BBIck81to VegF are described. The BCE103-Ink2-2BBIck81 fusion protein wasproduced in B. subtilis as described in Example 2. The fusion proteinwas purified, and the BBI trypsin inhibitory activity was increased bytreatment with βME and oxidized glutathione as described in Example 3.The fusion protein was cleaved by BsMpr protease (See, Example 4) andthe free 2BBIck81 was purified from the BCE103 catalytic domain by ionexchange chromatography using a Q-Sepharose column.

Briefly, after cleavage, the pH of the cleaved sample was adjusted to5.5, the sample was then loaded onto the column (equilibrated with 25 mMMES pH 5.5). The free 2BBIck81 was washed through the column using 25 mMsodium acetate pH 5.0 while the BCE103 catalytic core remained bound tothe resin. The 2BBIck81 fraction was concentrated by ultrafiltration andanalyzed using an electrochemiluminescence (ECL) based binding assay(BioVeris). The Anti-VegF antibody (Santa Cruz) and VegF (PeproTech)were labeled with the electrochemiluminescent dye and biotin,respectively, as described by the manufacturer (BioVeris). All materialswere in Dulbecco's PBS (Mediatech) supplemented with 0.1% TWEEN®-80. Aninitial dilution series of Anti-VegF antibody (125, 250 and 500 ng/ml)and VegF (100, 150, 200 and 250 ng/ml) were tested in the binding assayto determine the concentrations of each that would give a robust ECLsignal.

For testing 2BBIck81 binding, 50 μL of 500 ng/ml ECL labeled Anti-VegFantibody, 50 μL of 250 ng/ml biotinylated VegF and 100 μL 2BBIck81(series of 12.5, 15, 31.25, 62.5, 125, 250 or 500 ng/ml) were incubatedat room temperature for 2 h with shaking. Then, 50 μL of 0.2 mg/mlstreptavidin coated beads were added and the reaction was incubated atroom temperature for 30 minutes. The ECL signal was measured using aBioVeris M8/384 Analyzer as described by the manufacturer (BioVeris). Asshown in FIG. 6, the ECL signal decreased as increasing concentrationsof 2BBIck81 displaced more of the labeled Anti-VegF antibody bound toVegF attached to the magnetic beads.

Thus, the CK37281 peptide when grafted onto the chymotrypsin inhibitoryloop of BBI (2BBIck81) competed with the Anti-VegF antibody for bindingto VegF at micromolar concentrations. In fact, 2BBIck81 competed forVegF binding better than the synthesized CK37281 peptide itself (See,FIG. 6). The CK37281 peptide inserted into the trypsin inhibitory loop,1BBIck81, also competed with the Anti-VegF antibody in the BioVerisassay. Thus, BBI was found to be useful as a scaffold to present activebinding peptides selected by various screening methods.

Example 6 Use of Alternative Fusion Partners for the Production of2BBIck81

In this Example, experiments conducted to evaluate alternative fusionpartners are described. The DNA sequence of the oligonucleotide primersused to amplify the dsbC gene (E. coli) from pET-40b(+) are providedbelow. These primers generate a BssHII site at the 5′ end and a BamHI atthe 3′ end for cloning into p2JM103-Gen4-2BBIck81.

DsbCBBI-F (SEQ ID NO: 181)AACATGAGCGCGCAGGCTGATGACGCGGCAATTCAACAAACGTTAG DsbCBBI-R (SEQ ID NO:182) TCGTCTGGATCCGGTATGGGATCATTGTTGTCACCAGAACCACTAGTTGATCCTTTACCGCTGGTCATTTTTTGGTG

The DNA sequences of the oligonucleotides that were annealed together tomake a cassette (Alw44I-BamHI) for fusing the P. mendocina cutinase geneto BBI with Linker 2, are provided below.

CutinaseBBI+ (SEQ ID NO: 183)TGCACTTCTCTGCTTTGGTCTGTTGAACGCAGAGGTCTTGACAACAATGA TCCTATTCCGCutinaseBBI− (SEQ ID NO: 184)GATCCGGAATAGGATCATTGTTGTCAAGACCTCTGCGTTCAACAGACCAA AGCAGAGAAG

Because the BBI moiety has seven disulfide bonds, it is contemplatedthat higher titers of active BBI will be obtained using fusion proteinsother than the BCE103 cellulase catalytic domain. For example, in someembodiments, compositions such as thiol-disulfide oxidoreductases and/orprotein disulfide isomerases find use as fusion proteins to help producecorrectly folded BBI moieties. In this embodiment, no additionalactivation step is needed under most circumstances. In additionalembodiments, other proteins produced at high titers in B. subtilis alsofind use as fusion partners. For example, the thermostable proteindisulfide isomerase from the fungus Humicola insolens (hiPDI) has beenused as a fusion partner to produce the light chain of immunoglobulin G(2 disulfides) in Bacillus brevis (See, Kajino et al., Appl. Env.Microbiol., 66:638-642 [2000]).

To determine whether hiPDI could be a better fusion partner than BCE103for the production of BBI, this hiPDI gene was synthesized (DNA2.0) andcloned into the expression vector, p2JM103-Ink2-2BBIck81 (See, Example4) as a BssHII-SacI fragment. In designing the synthetic gene, codonsoccurring with high frequency in highly expressed B. subtilis genes wereselected except in cases where restriction sites were introduced ordeleted. In the final construction, the N-terminus of the mature hiPDIgene was fused to the AprE signal sequence and the C-terminus was fusedto a linker with an Enteropeptidase cleavage site (Kajino et al., Appl.Env. Microbiol., 66:638-642 [2000]), which in turn was fused to 2BBIck81(See, FIG. 7). This expression vector, p2JM-PDI-EK-2BBIck81, was used totransform B. subtilis BG6006 and the production of the fusion proteinwas determined in MBD medium (as described in Example 1) with or without2 mM BME added 14 h after inoculation.

As determined by SDS-PAGE gels, the production of the PDI-2BBIck81fusion protein was typically somewhat less than the BCE-2BBck81 grownunder identical conditions. The BBI titers (trypsin inhibition) measuredfrom the PDI-2BBIck81 cell free supernatants were also typically lessthan the BCE-2BBIck81 fusion. As with fusions to BCE103, the measuredactivities of BBI when fused to PDI were higher when grown in 2 mM BMEand the BBI activity was increased by the addition of BME to the cellfree supernatants after growth when grown in BME free medium (asdescribed in Example 3). Thus, the thiol-disulfide oxidoreductaseactivity of PDI does not seem to significantly improve the titers ofactive 2BBIck81 in the fusion protein or obviate the need for activationof the BBI molecule.

In order to increase the reduction potential of the fusion protein,which was contemplated to improve the BBI titers during growth, DsbCfrom Escherichia coliwas used as a fusion partner for 2BBIck81. The dsbCgene was amplified by PCR using Herculase Enhanced DNA polymerase asdescribed by the manufacturer (Stratagene) using DsbCBBI-F and DsbCBBI-Ras primers (sequences shown above) and pET-40b(+) (Novagen) as atemplate. The isolated PCR fragment was cloned into the vectorp2JM103-Gen4-2BBIck81 (See, Example 4) as a BssHII-BamHI fragment. Thecorrect sequence of the fusion gene was verified by DNA sequencing. Inthis case, the titers of the DsbC-2BBIck81 fusion protein weresignificantly lower than the BCE-2BBIck81 fusion protein as judged onSDS-PAGE gels and the titers of the active 2BBIck81 measured by trypsininhibition were much lower as well.

Other proteins that are produced at high titers in B. subtilis find useas fusion partners for the production of BBI. One such protein is thecutinase from Pseudomonas mendocina, which has been expressed at hightiters utilizing the aprE promoter from B. subtilis (See e.g., U.S. Pat.No. 5,429,950, herein incorporated by reference). The aprE-cutinase genefusion as an EcoRI-Alw44I fragment (from pAK-15) was ligated with anAlw44I-BamHI linker oligonucleotide cassette (See, sequence above) intothe p2JM103-Ink2-2BBIck81 (See, Example 4) that had been cut with EcoRIand BamHI. This cutinase-linker2-2BBIck81 expression vector (See, FIG. 8for the EcoRI-BamHI aprE-cutinase-linker2 sequence) was used totransform B. subtilis BG6006 cells and the fusion protein was producedin MBD medium as described previously for the other fusion proteins(See, Example 1). In this case, the cutinase-linker2-2BBIck81 fusionprotein was not the major band observed on SDS-PAGE gels and themeasured lipase titers (as measured using the methods provided in U.S.Pat. No. 5,429,950) and BBI titers were much less (ca. 20 fold) thanfound with the BCE-2BBIck81 fusion protein. Also, the BBI titers in thecutinase fusion protein were not improved significantly when 3 mM □MEwas added to the growth medium. Thus, the highest titers of active2BBIck81 was consistently obtained by activation of the BCE-2BBIck81fusion protein. Nonetheless, it is contemplated that various fusionpartners will find use in the present invention.

Example 7 Expression of Truncated BBI-AV(BBIt-AV; SEQ ID NO:187)

This Example describes experiments that were performed to improve theproduction of the 2BBIck81 BBI protein (BBI-AV; SEQ ID NO:186)

DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSEDDKEN(SEQ ID NO:18).

The Examples above and the journal article of Vogtentanz et al., 2007(Protein Expr Purif 55:40-52 [2007]) describe a method for producing afusion protein that comprises the soybean Bowman-Birk protease inhibitor(BBI) fused to the C-terminus of the BCE103 cellulase catalytic domain(BCE). As described above, this system has been used to produce BBImolecules with various variant peptides replacing the trypsin and/orchymotrypsin inhibitory loops. For example, a BBI molecule, BBI-AV (SEQID NO:186) containing the VEGF binding peptide, CK37281 (ACYNLYGWTC; SEQID NO:9), that replaces the native chymotrypsin inhibitory loop(ICALSYPAQC; SEQ ID NO:388) was produced, purified and shown to competein an electrochemiluminescent (ECL) based binding assay (BioVeris) witha monoclonal antibody for binding to VEGF. However, the recovered yieldof the variant BBI-AV was less than that of the wild-type BBI mostlikely due to an initial higher percentage of molecules with incorrectlyformed disulfide bonds.

A truncated form of the BBI-AV of SEQ ID NO:186 (i.e. BBIt-AV of SEQ IDNO:187)DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE (SEQID NO:187), which lacks the 5 C-terminal amino acids of SEQ ID NO:186was shown to bind better than the full-length molecule (i.e. untruncatedmolecule) to VEGF in an ECL (BioVeris) assay using a labeled monoclonalantibody against VEGF as a competitor. The C-truncated version ofBBI-AV, called BBIt-AV (SEQ ID NO:187), can be produced during thepurification process by trimming the C-terminus with GluBL protease(glutamyl endopeptidase I from Bacillus licheniformis). Alternatively,the BBIt-AV can be obtained by introducing a stop codon using anengineered oligonucleotide cassette as follows.

The following BBI-trunc+ and BBI-trunc− oligonucleotides were annealedto generate an oligonucleotide cassette

BBI-trunc+ (SEQ ID NO: 188)5′-TCGACATCACGGACTTCTGCTATGAACCATGTAAACCAAGCGAGTAA A and BBI-trunc− (SEQID NO: 189) 5′-AGCTTTTACTCGCTTGGTTTACATGGTTCATAGCAGAAGTCCGTGAT G

The oligonucleotide cassette was then ligated into the SalI and HindIIIsites of the expression vector p2JM103-Ink2-2BBIck81, and the correctsequence was verified and the resulting expression vector was calledp2JM103-Ink2-BBIt-AV.

To improve the expression, a polynucleotide sequence (SEQ ID NO:190gctggtaaa) encoding the first three amino acids of the AprE pro-peptide,AGK (SEQ ID NO:191) (U.S. Pat. No. 5,429,950), was inserted between theend of the AprE signal sequence and the start of the mature BCE103cellulase using a QuikChange® site-directed mutagenesis kit(Stratagene). The site directed mutagenesis was performed essentially asdescribed by the manufacturer using p2JM103-Ink2-BBIt-AV as a templatewith the oligonucleotide primers:

BCE-AGK-F: (SEQ ID NO: 192) 5′-GCGCGCAGGCAGCTGGTAAAGATGATTATTCAGTTGTAGAand BCE-AGK-R: (SEQ ID NO: 193)5′-AATAATCATCTTTACCAGCTGCCTGCGCGCTCATGTTGCT

The correct insertion was verified by DNA sequencing and the resultingexpression vector was called p2JMagk103-Ink2-BBIt-AV (SEQ ID NO:194).

(SEQ ID NO: 194) GAATTCTCCATTTTCTTCTGCTATCAAAATAACAGACTCGTGATTTTCCAAACGAGCTTTCAAAAAAGCCTCTGCCCCTTGCAAATCGGATGCCTGTCTATAAAATTCCCGATATTGGTTAAACAGCGGCGCAATGGCGGCCGCATCTGATGTCTTTGCTTGGCGAATGTTCATCTTATTTCTTCCTCCCTCTCAATAATTTTTTCATTCTATCCCTTTTCTGTAAAGTTTATTTTTCAGAATACTTTTATCATCATGCTTTGAAAAAATATCACGATAATATCCATTGTTCTCACGGAAGCACACGCAGGTCATTTGAACGAATTTTTTCGACAGGAATTTGCCGGGACTCAGGAGCATTTAACCTAAAAAAGCATGACATTTCAGCATAATGAACATTTACTCATGTCTATTTTCGTTCTTTTCTGTATGAAAATAGTTATTTCGAGTCTCTACGGAAATAGCGAGAGATGATATACCTAAATAGAGATAAAATCATCTCAAAAAAATGGGTCTACTAAAATATTATTCCATCTATTACAATAAATTCACAGAATAGTCTTTTAAGTAAGTCTACTCTGATTTTTTTAAAAGGAGAGGGTAAAGAGTGAGAAGCAAAAAATTGTGGATCAGCTTGTTGTTTGCGTTAACGTTAATCTTTACGATGGCGTTCAGCAACATGAGCGCGCAGGCAGCTGGTAAAGATGATTATTCAGTTGTAGAGGAACATGGGCAACTAAGTATTAGTAACGGTGAATTAGTCAATGAACGAGGCGAACAAGTTCAGTTAAAAGGGATGAGTTCCCATGGTTTGCAATGGTACGGTCAATTTGTAAACTATGAAAGCATGAAATGGCTAAGAGATGATTGGGGAATAACTGTATTCCGAGCAGCAATGTATACCTCTTCAGGAGGATATATTGACGATCCATCAGTAAAGGAAAAAGTAAAAGAGACTGTTGAGGCTGCGATAGACCTTGGCATATATGTGATCATTGATTGGCATATCCTTTCAGACAATGACCCGAATATATATAAAGAAGAAGCGAAGGATTTCTTTGATGAAATGTCAGAGTTGTATGGAGACTATCCGAATGTGATATACGAAATTGCAAATGAACCGAATGGTAGTGATGTTACGTGGGACAATCAAATAAAACCGTATGCAGAAGAAGTGATTCCGGTTATTCGTGACAATGACCCTAATAACATTGTTATTGTAGGTACAGGTACATGGAGTCAGGATGTCCATCATGCAGCCGATAATCAGCTTGCAGATCCTAACGTCATGTATGCATTTCATTTTTATGCAGGAACACATGGACAAAATTTACGAGACCAAGTAGATTATGCATTAGATCAAGGAGCAGCGATATTTGTTAGTGAATGGGGGACAAGTGCAGCTACAGGTGATGGTGGTGTGTTTTTAGATGAAGCACAAGTGTGGATTGACTTTATGGATGAAAGAAATTTAAGCTGGGCCAACTGGTCTCTAACGCATAAGGATGAGTCATCTGCAGCGTTAATGCCAGGTGCAAATCCAACTGGTGGTTGGACAGAGGCTGAACTATCTCCATCTGGTACATTTGTGAGGGAAAAAATAAGAGAATCAGCATCTGACAACAATGATCCCATACCGGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTTTTTGCGTCGACATCACGGACTTCTGCTATGAACCATGTAAACCAAGCGAGTAAAAGCTTAACTCGAGGTTAACAGAGGACGGATTTCCTGAAGGAAATCCGTTTTTTTATTTTTAATTAAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCTGGAGCTGTAATATAAAAACCTTCTTCAACTAACGGGGCAGGTTAGTGACATTAGAAAACCGACTGTAAAAAGTACAGTCGGCATTATCTCATATTATAAAAGCCAGTCATTAGGCCTATCTGACAATTCCTGAATAGAGTTCATAAACAATCCTGCATGATAACCATCACAAACAGAATGATGTACCTGTAAAGATAGCGGTAAATATATTGAATTACCTTTATTAATGAATTTTCCTGCTGTAATAATGGGTAGAAGGTAATTACTATTATTATTGATATTTAAGTTAAACCCAGTAAATGAAGTCCATGGAATAATAGAAAGAGAAAAAGCATTTTCAGGTATAGGTGTTTTGGGAAACAATTTCCCCGAACCATTATATTTCTCTACATCAGAAAGGTATAAATCATAAAACTCTTTGAAGTCATTCTTTACAGGAGTCCAAATACCAGAGAATGTTTTAGATACACCATCAAAAATTGTATAAAGTGGCTCTAACTTATCCCAATAACCTAACTCTCCGTCGCTATTGTAACCAGTTCTAAAAGCTGTATTTGAGTTTATCACCCTTGTCACTAAGAAAATAAATGCAGGGTAAAATTTATATCCTTCTTGTTTTATGTTTCGGTATAAAACACTAATATCAATTTCTGTGGTTATACTAAAAGTCGTTTGTTGGTTCAAATAATGATTAAATATCTCTTTTCTCTTCCAATTGTCTAAATCAATTTTATTAAAGTTCATTTGATATGCCTCCTAAATTTTTATCTAAAGTGAATTTAGGAGGCTTACTTGTCTGCTTTCTTCATTAGAATCAATCCTTTTTTAAAAGTCAATATTACTGTAACATAAATATATATTTTAAAAATATCCCACTTTATCCAATTTTCGTTTGTTGAACTAATGGGTGCTTTAGTTGAAGAATAAAAGACCACATTAAAAAATGTGGTCTTTTGTGTTTTTTTAAAGGATTTGAGCGTAGCGAAAAATCCTTTTCTTTCTTATCTTGATAATAAGGGTAACTATTGCCGGATCGTCCTCAGGAGTAGGCGACATCGCTAAATAATGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT

The p2JMagk103-Ink2-BBIt-AV expression vector was used to transformbacterial cells to express BBIt-AV fused to the BCE protein (SEQ IDNO:195)

(SEQ ID NO: 195) AGKDDYSVVEEHGQLSISNGELVNERGEQVQLKGMSSHGLQWYGQFVNYESMKWLRDDWGITVFRAAMYTSSGGYIDDPSVKEKVKETVEAAIDLGIYVIIDWHILSDNDPNIYKEEAKDFFDEMSILYGDYPNVIYEIANEPNGSDVTWDNQIKPYAEEVIPVIRDNDPNNIVIVGTGTWSQDVHHAADNQLADPNVMYAFHFYAGTHGQNLRDGVDYALDQGAAIFVSEWGTSAATGDGGVFLDEAQVWIDFMDERNLSWANWSLTHKDESSAALMPGANPTGGWTEAELSPSGTFVREKIRESASDNNDPIPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLGWTCFCVDITDFCYEPCKPSE

Example 8 BBIt-AV Site Saturation Libraries

This example describes experiments that were performed to generatemodified variant BBPIs derived from the unmodified BBIt-AV byconstructing site saturation libraries comprising substitutions at eachof the 39 amino acid positions of the BBIt-AV molecule (SEQ ID NO:187)shown in FIG. 9.

BBIt-AV site-saturation libraries were created essentially as describedby Amin et al. (Biotechniques 35: 1134-1140, [2003]) using a modifiedversion of the QuikChange multi site-directed mutagenesis (QCMS) kitfrom Stratagene (La Jolla, Calif.) as follows. Overlapping forward andreverse primers with the NNS codon in the middle and 17-20 flankingbases were designed for each chosen site, and the sequences for eachforward (F) and reverse (R) primer at each of the 66 amino acidpositions in the BBI-AV molecule are shown in Table 1. Each mutagenesisreaction contained 50-100 ng template plasmid (p2JMagk103-Ink2-BBIt-AV;SEQ ID NO:194), 0.5 μl forward primer (25 μM), 0.5 μl reverse primer (25μM), 1 μl dNTP's (QCMS kit), 2.5 μl 10×QCMS reaction buffer, 18.5 μldeionized water, and 1 μl of enzyme blend (QCMS kit), for a total volumeof 25 μl. For BBI-AV libraries at residues 10, 11, 13 and 25, only 1 μLof the forward primer (25 □M) was used without the reverse primer. Forlibraries at residues 6, 37, 38, 50 and 53, the mutagenesis reaction wascarried out with 0.5 μl of the forward mutagenic primer in combinationwith another primer (5′-CTATGCGGCATCAGAGCAGATTGTAC; SEQ ID NO:328complementary to a sequence in the vector. For the mutagenesis reaction,the thermocycler program used was 1 cycle at 95° for 2 min., followed by29 cycles of 95° C. for 30 sec, 55° C. for 1 min., and 68° C. for 10minutes (MJ Research thermocycler). The template DNA was digested by theaddition of 0.5-1 μl DpnI (from the QCMS kit) and incubation at 37° C.for 1-4 hours, followed by another addition of 0.5-1 μl DpnI and anotherincubation at 37° C. for 1-4 hours. The template DNA was obtained fromE. coli dam⁺ strains, such as TOP10 (Invitrogen, Carlsbad, Calif., USA),to ensure that it would be susceptible to Dpn I digestion.

For efficient transformation of B. subtilis, the DNA in each mutagenesisreaction was amplified by rolling circle amplification (RCA) using theTempliphi kit from Amersham. Specifically, 1 μL of the undiluted QCMSreaction was mixed with 5 μL of sample buffer from the Templiphi kit.The mixture was heated for 3 minutes at 95° C. to denature the DNA. Thereaction was placed on ice to cool for 2 minutes. Then, 5 μL of reactionbuffer and 0.2 μL of phi29 polymerase were added to the reaction andincubated at 30° C. in a MJ Research thermocycler for 5 hours. Finally,the phi29 enzyme in the reaction was heat inactivated by incubation at65° C. for 10 min in a MJ Research thermocycler. The amplified DNA wasdiluted 10-100 fold and 1 μL was used to transform 100 μL of competentB. subtilis BG6006 cells (Vogtentanz et al., 2007). Aliquots of 25 μL or75 μL of the transformation mixture were plated on Luria Agar platessupplemented with 5 μg/ml chloramphenicol. After growth, individualtransformants were picked to inoculate 200 μl of Luria brothsupplemented with 5 μg/ml chloramphenicol in each well of a 96-wellmicrotiter plate. The cultures were grown in a humidified and aeratedbox at 37° C. and 270 rpm (Innova 4230 incubator, New BrunswickScientific). After growth, 80 μl of 50% glycerol were added to each welland mixed. A duplicate plate was made by removing 140 μl of the culturefrom each well and placing it in a new microtiter plate. Both plateswere stored at −80° C. One plate was saved as a master and the other wassubmitted for DNA sequencing (Cogenics, Houston, Tex.) of the individualmutants. For DNA sequencing, an overnight culture of each colony grownin a microtiter plate was diluted 10-40 fold and 2 μL was used in a PCRreaction with Amersham Biosciences Ready-to-go PCR beads and the primersBBI-PCR-F and BBI-PCR-R. The thermo-cycling conditions were one cycle at95° C. for 5 minutes, followed by 30 cycles of 95° C. for 1 minute, 55°C. for 1 minute and 72° C. for 1 minute, and a final cycle at 72° C. for7 minutes. The PCR products were treated with Exonuclease I and shrimpalkaline phosphatase (EXOSAP-IT, Amersham Biosciences) or they werecolumn purified before sequencing with the M13 reverse primer.

TABLE 1 NNS mutagenesis primers Primer name Primer Sequence SEQ ID NO:Forward Primers BBI1F GATCCCATACCGGATCCANNSGATGAG 196 AGCTCTAAACC BBI2FCCATACCGGATCCAGACNNSGAGAGCT 197 CTAAACCCTG BBI3FCATACCGGATCCAGACGATNNSAGCTC 198 TAAACCCTGTTG BBI4FCGGATCCAGACGATGAGNNSTCTAAAC 199 CTGTTGCGATC BBI5FGATCCAGACGATGAGAGCNNSAAACCC 200 TGTTGCGATCAATG BBI6FCAGACGATGAGAGCTCTNNSCCCTGTT 201 GCGATCAATG BBI7FGACGATGAGAGCTCTAAANNSTGTTGC 202 GATCAATGCGC BBI8FGATGAGAGCTCTAAACCCNNSTGCGAT 203 CAATGCGCATG BBI9F GAGAGCTCTAAACCCTGTNN204 SGATCAATGCGCATGTAC BBI10F GCTCTAAACCCTGTTGCNNSCAATGCG 205CATGTACGAAATC BBI11F CTAAACCCTGTTGCGATNNSTGCGCAT 206 GTACGAAATC BBI12FCTAAACCCTGTTGCGATCAANNSGCAT 207 GTACGAAATCAAATC BBI13FCCTGTTGCGATCAATGCNNSTGTACGA 208 AATCAAATCC BBI14FGTTGCGATCAATGCGCANNSACGAAAT 209 CAAATCCTCC BBI15FGCGATCAATGCGCATGTNNSAAATCAA 210 ATCCTCCACAG BBI16FGATCAATGCGCATGTACGNNSTCAAAT 211 CCTCCACAGTG BBI17FCAATGCGCATGTACGAAANNSAATCCT 212 CCACAGTGTCG BBI18FGCGCATGTACGAAATCANNSCCTCCAC 213 AGTGTCGGTG BBI19FCATGTACGAAATCAAATNNSCCACAGT 214 GTCGGTGTTC BBI20FGTACGAAATCAAATCCTNNSCAGTGTC 215 GGTGTTCCGATAT BBI21FCGAAATCAAATCCTCCANNSTGTCGGT 216 GTTCCGATATG BBI22FGAAATCAAATCCTCCACAGNNSCGGTG 217 TTCCGATATGCG BBI23FCAAATCCTCCACAGTGTNNSTGTTCCG 218 ATATGCGTCTG BBI24FCAAATCCTCCACAGTGTCGGNNSTCCG 219 ATATGCGTCTGAATTC BBI25FCTCCACAGTGTCGGTGTNNSGATATGC 220 GTCTGAATTC BBI26FCACAGTGTCGGTGTTCCNNSATGCGTC 221 TGAATTCCTG BBI27FCAGTGTCGGTGTTCCGATNNSCGTCTG 222 AATTCCTGTCATAG BBI28FGTCGGTGTTCCGATATGNNSCTGAATT 223 CCTGTCATAG BBI29FGGTGTTCCGATATGCGTNNSAATTCCT 224 GTCATAGTGC BBI30FGTTCCGATATGCGTCTGNNSTCCTGTC 225 ATAGTGCCTG BBI31FCCGATATGCGTCTGAATNNSTGTCATA 226 GTGCCTGCAAAAG BBI32FGATATGCGTCTGAATTCCNNSCATAGT 227 GCCTGCAAAAG BBI33FATATGCGTCTGAATTCCTGTNNSAGTG 228 CCTGCAAAAGCTG BBI34FGTCTGAATTCCTGTCATNNSGCCTGCA 229 AAAGCTGCGC BBI35FCTGAATTCCTGTCATAGTNNSTGCAAA 230 AGCTGCGCATG BBI36FGAATTCCTGTCATAGTGCCNNSAAAAG 231 CTGCGCATGTTATAA BBI37FCCTGTCATAGTGCCTGCNNSAGCTGCG 232 CATGTTATAAC BBI38FGTCATAGTGCCTGCAAANNSTGCGCAT 233 GTTATAACCTG BBI39FCATAGTGCCTGCAAAAGCNNSGCATGT 234 TATAACCTGTAC BBI40FGTGCCTGCAAAAGCTGCNNSTGTTATA 235 ACCTGTACGG BBI41FCCTGCAAAAGCTGCGCANNSTATAACC 236 TGTACGGGTG BBI42FGCAAAAGCTGCGCATGTNNSAACCTGT 237 ACGGGTGGAC BBI43FCAAAAGCTGCGCATGTTATNNSCTGTA 238 CGGGTGGACCTG BBI44FGCTGCGCATGTTATAACNNSTACGGGT 239 GGACCTGTTTTTG BBI45FGCGCATGTTATAACCTGNNSGGGTGGA 240 CCTGTTTTTG BBI46FCATGTTATAACCTGTACNNSTGGACCT 241 GTTTTTGCGTC BBI47FGTTATAACCTGTACGGGNNSACCTGTT 242 TTTGCGTCGAC BBI48FGTTATAACCTGTACGGGTGGNNSTGTT 243 TTTGCGTCGACATC BBI49FATAACCTGTACGGGTGGACCNNSTTTT 244 GCGTCGACATCAC BBI50FCTGTACGGGTGGACCTGTNNSTGCGTC 245 GACATCACGGAC BBI51FGTACGGGTGGACCTGTTTTNNSGTCGA 246 CATCACGGACTTC BBI52FGGTGGACCTGTTTTTGCNNSGACATCA 247 CGGACTTCTG BBI53FGGACCTGTTTTTGCGTCNNSATCACGG 248 ACTTCTGCTATG BBI54FCCTGTTTTTGCGTCGACNNSACGGACT 249 TCTGCTATGAAC BBI55FGTTTTTGCGTCGACATCNNSGACTTCT 250 GCTATGAACC BBI56FGTTTTTGCGTCGACATCACGNNSTTCT 251 GCTATGAACCATG BBI57FGCGTCGACATCACGGACNNSTGCTATG 252 AACCATGTAAAC BBI58FGTCGACATCACGGACTTCNNSTATGAA 253 CCATGTAAACC BBI59FGACATCACGGACTTCTGCNNSGAACCA 254 TGTAAACCAAG BBI60FCATCACGGACTTCTGCTATNNSCCATG 255 TAAACCAAGCGAG BBI61FCGGACTTCTGCTATGAANNSTGTAAAC 256 CAAGCGAGTAAAA BBI62FGACTTCTGCTATGAACCANNSAAACCA 257 AGCGAGTAAAAG BBI63FCTTCTGCTATGAACCATGTNNSCCAAG 258 CGAGTAAAAGCTTAA BBI64FGCTATGAACCATGTAAANNSAGCGAGT 259 AAAAGCTTAAC BBI65FCTATGAACCATGTAAACCANNSGAGTA 260 AAAGCTTAACTC BBI66FGAACCATGTAAACCAAGCNNSTAAAAG 261 CTTAACTCGAG Reverse Primers BBI1RGGTTTAGAGCTCTCATCSNNTGGATCC 262 GGTATGGGATC BBI2RCAGGGTTTAGAGCTCTCSNNGTCTGGA 263 TCCGGTATGG BBI3RCAACAGGGTTTAGAGCTSNNATCGTCT 264 GGATCCGGTATG BBI4RGATCGCAACAGGGTTTAGASNNCTCAT 265 CGTCTGGATCCG BBI5RCATTGATCGCAACAGGGTTTSNNGCTC 266 TCATCGTCTGGATC BBI6RCATTGATCGCAACAGGGSNNAGAGCTC 267 TCATCGTCTG BBI7RGCGCATTGATCGCAACASNNTTTAGAG 268 CTCTCATCGTC BBI8RCATGCGCATTGATCGCASNNGGGTTTA 269 GAGCTCTCATC BBI9RGTACATGCGCATTGATCSNNACAGGGT 270 TTAGAGCTCTC BBI10RGATTTCGTACATGCGCATTGSNNGCAA 271 CAGGGTTTAGAGC BBI11RGATTTCGTACATGCGCASNNATCGCAA 272 CAGGGTTTAG BBI12RGATTTGATTTCGTACATGCSNNTTGAT 273 CGCAACAGGGTTTAG BBI13RGGATTTGATTTCGTACASNNGCATTGA 274 TCGCAACAGG BBI14RGGAGGATTTGATTTCGTSNNTGCGCAT 275 TGATCGCAAC BBI15RCTGTGGAGGATTTGATTTSNNACATGC 276 GCATTGATCGC BBI16RCACTGTGGAGGATTTGASNNCGTACAT 277 GCGCATTGATC BBI17RCGACACTGTGGAGGATTSNNTTTCGTA 278 CATGCGCATTG BBI18RCACCGACACTGTGGAGGSNNTGATTTC 279 GTACATGCGC BBI19RGAACACCGACACTGTGGSNNATTTGAT 280 TTCGTACATG BBI20RATATCGGAACACCGACACTGSNNAGGA 281 TTTGATTTCGTAC BBI21RCATATCGGAACACCGACASNNGGAGGA 282 TTTGATTTCG BBI22RCGCATATCGGAACACCGSNNCTGTGGA 283 GGATTTGATTTC BBI23RCAGACGCATATCGGAACASNNACACTG 284 TGGAGGATTTG BBI24RGAATTCAGACGCATATCGGASNNCCGA 285 CACTGTGGAGGATTTG BBI25RGAATTCAGACGCATATCSNNACACCGA 286 CACTGTGGAG BBI26RCAGGAATTCAGACGCATSNNGGAACAC 287 CGACACTGTG BBI27RCTATGACAGGAATTCAGACGSNNATCG 288 GAACACCGACACTG BBI28RCTATGACAGGAATTCAGSNNCATATCG 289 GAACACCGAC BBI29RGCACTATGACAGGAATTSNNACGCATA 290 TCGGAACACC BBI30RCAGGCACTATGACAGGASNNCAGACGC 291 ATATCGGAAC BBI31RCTTTTGCAGGCACTATGACASNNATTC 292 AGACGCATATCGG BBI32RCTTTTGCAGGCACTATGSNNGGAATTC 293 AGACGCATATC BBI33RCAGCTTTTGCAGGCACTSNNACAGGAA 294 TTCAGACGCATAT BBI34RGCGCAGCTTTTGCAGGCSNNATGACAG 295 GAATTCAGAC BBI35RCATGCGCAGCTTTTGCASNNACTATGA 296 CAGGAATTCAG BBI36RTTATAACATGCGCAGCTTTTSNNGGCA 297 CTATGACAGGAATTC BBI37RGTTATAACATGCGCAGCTSNNGCAGGC 298 ACTATGACAGG BBI38RCAGGTTATAACATGCGCASNNTTTGCA 299 GGCACTATGAC BBI39RGTACAGGTTATAACATGCSNNGCTTTT 300 GCAGGCACTATG BBI40RCCGTACAGGTTATAACASNNGCAGCTT 301 TTGCAGGCAC BBI41RCACCCGTACAGGTTATASNNTGCGCAG 302 CTTTTGCAGG BBI42RGTCCACCCGTACAGGTTSNNACATGCG 303 CAGCTTTTGC BBI43RCAGGTCCACCCGTACAGSNNATAACAT 304 GCGCAGCTTTTG BBI44RCAAAAACAGGTCCACCCGTASNNGTTA 305 TAACATGCGCAGC BBI45RCAAAAACAGGTCCACCCSNNCAGGTTA 306 TAACATGCGC BBI46RGACGCAAAAACAGGTCCASNNGTACAG 307 GTTATAACATG BBI47RGTCGACGCAAAAACAGGTSNNCCCGTA 308 CAGGTTATAAC BBI48RGATGTCGACGCAAAAACASNNCCACCC 309 GTACAGGTTATAAC BBI49RGTGATGTCGACGCAAAASNNGGTCCAC 310 CCGTACAGGTTAT BBI50RGTCCGTGATGTCGACGCASNNACAGGT 311 CCACCCGTACAG BBI51RGAAGTCCGTGATGTCGACSNNAAAACA 312 GGTCCACCCGTAC BBI52RCAGAAGTCCGTGATGTCSNNGCAAAAA 313 CAGGTCCACC BBI53RCATAGCAGAAGTCCGTGATSNNGACGC 314 AAAAACAGGTCC BBI54RGTTCATAGCAGAAGTCCGTSNNGTCGA 315 CGCAAAAACAGG BBI55RGGTTCATAGCAGAAGTCSNNGATGTCG 316 ACGCAAAAAC BBI56RCATGGTTCATAGCAGAASNNCGTGATG 317 TCGACGCAAAAAC BBI57RGTTTACATGGTTCATAGCASNNGTCCG 318 TGATGTCGACGC BBI58RGGTTTACATGGTTCATASNNGAAGTCC 319 GTGATGTCGAC BBI59RCTTGGTTTACATGGTTCSNNGCAGAAG 320 TCCGTGATGTC BBI60RCTCGCTTGGTTTACATGGSNNATAGCA 321 GAAGTCCGTGATG BBI61RTTTTACTCGCTTGGTTTACASNNTTCA 322 TAGCAGAAGTCCG BBI62RCTTTTACTCGCTTGGTTTSNNTGGTTC 323 ATAGCAGAAGTC BBI63RTTAAGCTTTTACTCGCTTGGSNNACAT 324 GGTTCATAGCAGAAG BBI64RGTTAAGCTTTTACTCGCTSNNTTTACA 325 TGGTTCATAGC BBI65RGAGTTAAGCTTTTACTCSNNTGGTTTA 326 CATGGTTCATAG BBI66RCTCGAGTTAAGCTTTTASNNGCTTGGT 327 TTACATGGTTC

The cultures in each master plate were thawed and a pin replicator wasused to inoculate a new sterile microtiter plate containing 150 μl ofLuria broth with 5 μg/ml chloramphenicol in each well. The plates weregrown for ˜10 hrs in a humidified and aerated box as described above.For controls, the cultures in two wells were replaced with BBI andBBI-AV cultures that had been grown concurrently in 15 ml culture tubes(5 ml of Luria broth with 5 μg/ml chloramphenicol). After the cultureswere grown, the plate was used to inoculate two duplicate 96-well filterplates (Millipore, MSGV2210) containing 150 μl of MBD medium (Vogtentanzet al., 2007) with 5 μg/ml chloramphenicol in each well by using a pinreplicator. The duplicate plates were grown for ˜14 hours (as above,with plastic spacers placed between the stacked plates to allow forefficient air flow around the plates) and then 2-mecaptoethanol wasadded to each well in one of the plates to a final concentration of 2mM. The plates were then grown for an additional ˜46 hours. The 96-wellfilter plates containing the culture broth (with or without added2-mecaptoethanol) were then assayed for BBI and BCE activity.

Example 9 Primary Screening of BBIt-AV Variants: BBI and BCE ActivityAssays to Determine Variants Having the Highest BBIt-AV:BCE ActivityRatio

In this Example, methods are provided for assessing the effect of theamino acid substitutions introduced into the modified variant BBIt-AVsgenerated according to the site-saturation methods. The trypsininhibitory activity of the modified BBIt-AV generated by thesite-saturation methods described above was measured and compared to thetrypsin inhibitory activity of the control wild-type BBI (SEQ ID NO:13;BBI-wt or sBBI) and the control unmodified BBIt-AV (SEQ ID NO:187). Itis not intended that the present invention be limited to these specificmethods, as other suitable methods find use.

Trypsin Inhibitory Assay: BBI Activity Determination

The relative concentration of active control and modified BBIt-AV's wasdetermined by a trypsin inhibitory assay using purified sBBI as astandard as described above. For some samples, the active BBIconcentration was measured by residual trypsin activity according to themethod described by Flecker (Flecker, P. FEBS Lett. 252:153-157 [1989];Vogtentanz et al. supra).

Libraries were analyzed using dilutions of cultures grown in 96-wellplates. Typically, the culture broth from each well was diluted (200fold) in Assay Buffer (100 mM Tris pH 8.6, 0.005% Tween 80) andtransferred to a second microtiter plate (CoStar 9017, Corning, Inc.,Corning, N.Y.). Then, 20 μl of the diluted samples were transferred to athird microtiter plate. A standard curve was created by adding 20, 10, 5or 2.5 μl of the diluted sBBI (SEQ ID NO:13) control culture to emptywells. Then, 80 μL of 50 ng/ml trypsin (bovine pancreatic trypsin,Worthington Biochemical Corp., Lakewood, N.J.) (prepared from a 1 mg/mLstock solution diluted into Assay Buffer) was added to each well of theplate. The reactions were mixed and incubated 15 min at roomtemperature. After the incubation, 100 μL of 0.5 mg/ml trypsin substrate(succinyl-Ala-Ala-Pro-Arg-para-nitroanilide, Bachem Bioscience, Inc.,King of Prussia, Pa.), diluted in Assay Buffer from a 100 mg/ml solutionin DMSO, was added to each well, mixed, and the absorbance at 405 nm wasmonitored for 15 min, with 1 time point recorded every 12 sec using aSpectra Max 250 (Molecular Devices). The data points were used todetermine the rate for each reaction. The standard curve was generatedby plotting the reaction rate versus the BBI volume and was fitted to afour-parameter equation. All data fitting was done using softwaresupplied by the manufacturer (Molecular Devices Corp., Sunnyvale,Calif.). The activity of each BBIt-AV variant was calculated from thestandard curve. Thus, the activities of all modified variant proteinswere determined relative to the activity in the sBBI control (BBI-WT)culture. If the majority of the data did not fall near the IC₅₀ of thestandard curve, a new dilution plate was made and assayed.

Cellulase Enzyme Activity Assay: BCE Assay

The concentration of BCE was determined by an activity assay usingpurified BCE as a standard as described above and by (Vogtentanz et al.2007). For the analysis of libraries grown in 96-well microtiter plates,20 μl of culture broth were transferred to each well of a new microtiterplate. A standard curve was created by adding 20, 10, 5 or 2.5 μl of thediluted BBI-WT control culture to these empty wells. Then, 180 μl of theBCE substrate (0.25 μg/ml in Assay Buffer), 4-nitrophenylβ-D-cellobioside (Sigma, St. Louis, Mo.), was added to the plate. Theplate was mixed and the absorbance at 405 nm monitored for 15 min, with1 time point recorded every 12 s using a Spectra Max 250 (MolecularDevices Corp., Sunnyvale, Calif.). The data points were used todetermine the rate for each reaction. The standard curve was generatedby plotting rate versus culture volume and was fit to a quadratic curve.The BCE concentration in each variant culture was determined relative tothe standard control culture.

Enzyme Activity Ratio: BBIt-AV:BCE Ratios

From the microtiter plate assay results, the BBIt-AV:BCE activity ratioof each well was determined by dividing the relative BBIt-AV trypsininhibitory activity values by the relative BCE activity values. The datawere sorted based on the BBIt-AV:BCE activity ratio. Modified variantswith the highest BBIt-AV:BCE activity ratios were selected and subjectedto a secondary screening described below.

Analysis of Enzyme Activity ratio BBIt-AV:BCE—Secondary Screening ofVariants

In this example, modified BBIt-AVs having improved BBIt-AV:BCE activityrelative to that of the control unmodified BBIt-AV when grown in thepresence or absence of a reducing agent (2-mercaptoethanol), asdetermined in the primary screen, were arrayed and assayed inquadruplicate in a secondary screen. Initially, only the polynucleotidesencoding each of the modified BBIt-AVs having the greatest BBIt-AV:BCEratio as determined in the secondary screen were sequenced. Later, allBBIt-AV clones were sequenced.

Quadruplicate Testing in Microtiter Plates—Secondary Screen

For each plate, twenty-two samples of cultures containing modifiedBBIt-AVs selected from the initial screen, and two samples of controlcultures containing sBBI and unmodified BBIt-AV, were grown for ˜10 hrsin 5 ml of Luria broth with 5 ppm chloramphenicol (37° C., 250 rpm) in15 ml culture tubes. Then, 150 μl of each of the 24 cultures werearrayed in quadruplicate in a 96-well microtiter plate. This arrayedplate was then used to inoculate duplicate filter plates (Millipore,MSGV2210) containing 150 μl of MBD medium with 5 μg/ml chloramphenicolper well by using a pin replicator. The plated cultures were grown (inthe presence and absence of 2-β-mercaptoethanol (βME), and theBBI-AV:BCE ratios were determined and analyzed as described above.

FIG. 10 shows an example of the effect of all possible 19 amino acidsubstitutions at position 29 of SEQ ID NO:187 on the trypsin inhibitoryactivity of the modified variant BBIt. The data show the ratio of BBIand BCE activities determined from quadruplicate measurements of BCE andtrypsin activities of the modified BBI-AVs having a single amino acidsubstitution at position 29 of SEQ ID NO:187.

Example 10 Selection for Improved Modified BBIt-AVs with Known AminoAcid Substitutions

In this example, site saturation libraries were constructed to generatea complete set of amino acid substitutions at sites identified in theinitial screen that resulted in BBIt-AVs having improved trypsininhibitory activity when compared to the trypsin inhibitory activity ofthe unmodified parent BBI-AV. In addition, a complete set of amino acidsubstitutions at sites that had not been previously substituted with allpossible 19 amino acids were generated by site-directed mutagenesis. Atotal of 39 site saturation libraries were generated and quadruplicateactivity measurements of BBI trypsin inhibitory activity and BCEactivity were analyzed to determine the single substitutions thatresulted in modified BBIt-AV molecules having trypsin inhibitoryactivity that was greater than that of the control BBI molecules i.e.sBBI and unmodified BBIt-AV. The positions of the amino acids that weresubstituted in the unmodified BBIt-AV are shown in FIG. 9.

Construction of a Second Set of Site-Saturation Libraries at Sites 18,38 and 61

First, to aid library construction, a synthetic gene was synthesized toreplace the EcoRI site with a SphI site between the region coding forthe trypsin inhibitory loop and the region encoding the chymotypsininhibitory loop in the 2BBIck81 coding region (the location of the EcoRIsite is shown in FIG. 3). The synthetic gene sequence is:5′-GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCGCTTGTACAAAATCAAACCCTCCACAATGTCGTTGTTCTGATATGCGTTTAAATAGCTGTCATTCTGCATGCAAATCATGTGCTTGCTATAACCTTTACGGTTGGACATGTTTCTGCGTCGACATCACTGACTTCTGCTATGAACCATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO:329). Thissynthetic gene was cloned into p2JMagk103-Ink2-BBIt-AV as aBamHI-HindIII fragment and the resulting vector was calledp2JMagk103-Ink2-BBIt-AVsph.

Site-saturation libraries were constructed at positions 18, 37 and 61using oligonucleotide cassettes as follows.

A site-saturation library at site 18 was constructed by annealingoligonucleotides5′-GTACAAAATCANNSCCTCCACAATGTCGTTGTTCTGATATGCGTTTAAATAGCTGTCATTCTGCATG(SEQ ID NO:330) and5′-CAGAATGACAGCTATTTAAACGCATATCAGAACAACGACATTGTGGAGGSNNTGATTTT (SEQ IDNO:331) and ligating the resulting cassette into the BsrGI and SphIsites of p2JMagk103-Ink2-BBI-AVsph.

A site-saturation library at site 37 was constructed by annealing theoligonucleotides5′-TCGACGCAGAAACATGTCCAACCGTAAAGGTTATAGCAAGCACATGASNNGCATG (SEQ IDNO:332) and 5′-CNNSTCATGTGCTTGCTATAACCTTTACGGTTGGACATGTTTCTGCG (SEQ IDNO:333) and ligating the resulting cassette into the SalI and SphI sitesof p2JMagk103-Ink2-BBI-AVsph.

The site-saturation library at position 61 was made by annealingoligonucleotide 5′-TCGACATCACTGACTTCTGCTATGAANNSTGTAAACCTTCTGAATAAA (SEQID NO:334) with the two oligonucleotides 5′-TTCATAGCAGAAGTCAGTGATG (SEQID NO:335) and 5′-AGCTTTTATTCAGAAGGTTTACA (SEQ ID NO:336) and ligatingthis cassette into the SalI and HindIII sites ofp2JMagk103-Ink2-BBIt-AVsph.

The ligation mixes were used to transform E. coli TOP10 cells(Invitrogen). Ninety six transformants were selected, and the amino acidsubstitution present in each clone was determined by DNA sequencing.Plasmids isolated from each single amino acid substitution were thenused to transform B. subtilis.

Generation of Amino Acid Substitutions by Site Directed Mutagenesis

Amino acid substitutions at selected sites were constructed by ligatingan oligonucleotide or an oligonucleotide cassette into the ofp2JMagk103-Ink2-BBI-AVsph vector using the appropriate restrictionsites. The substitutions, oligonucleotide sequences and restrictionsites used are shown in Table 2.

TABLE 2 Oligonucleotides for site directed muatagenesis Amino AcidSubstitution(s) Oligonucleotide Pairs Sequence(s) Restriction Sites A13Y5′-Phos-GTACAGTAGCATTGATCGCAACAAGGTT SacI & BsrGI TAGAGCT (SEQ ID NO:337) R23D, E 5′-Phos-GTACAAAATCAAACCCTCCACAATGTGA BsrGI & SphINTGTTCTGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 338)5′-CAGAATGACAGCTATTTAAACGCATATCAGAAC ANTCACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 339) S25M 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTATGGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 340)5′-CAGAATGACAGCTATTTAAACGCATATCCATAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 341) S25W 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTGGGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 342)5′-CAGAATGACAGCTATTTAAACGCATATCCCAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 343) S25A, V, G 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTGBAGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 344)5′-CAGAATGACAGCTATTTAAACGCATATCTVCAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 345) S25N, Q, K, H 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTMAWGATATGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 346)5′-CAGAATGACAGCTATTTAAACGCATATCWTKAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 347) M27W 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATTGGCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 348)5′-CAGAATGACAGCTATTTAAACGCCAATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 349) M27H 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATCACCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 350)5′-CAGAATGACAGCTATTTAAACGGTGATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 351) M27G, A, V, D 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATGNTCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 352)5′-CAGAATGACAGCTATTTAAACGANCATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 353) M27F, I 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATWTTCGTTTAAATAGCTGTCATTCTGC ATG (SEQ ID NO: 354)5′-CAGAATGACAGCTATTTAAACGAAWATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 355) L29I 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTATTAATAGCTGTCATTCTGC ATG (SEQ ID NO: 356)5′-CAGAATGACAGCTATTAATACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 357) L29D, E 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTGANAATAGCTGTCATTCTGC ATG (SEQ ID NO: 358)5′-CAGAATGACAGCTATTNTCACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 359) S31M 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTTTAAATATGTGTCATTCTGC ATG (SEQ ID NO: 360)5′-CAGAATGACACATATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 361) S31D, E, Y 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTTTAAATKAWTGTCATTCTGC ATG (SEQ ID NO: 362)5′-CAGAATGACAWTMATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 363) S31Q, H 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTTTAAATCANTGTCATTCTGC ATG (SEQ ID NO: 364)5′-CAGAATGACANTGATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 365) S34I 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTTTAAATAGCTGTCATATCGC ATG (SEQ ID NO: 366)5′-CGATATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 367) S34W 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTTTAAATAGCTGTCATTGGGC ATG (SEQ ID NO: 368)5′-CCCAATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 369) S34R, Q 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTTTAAATAGCTGTCATCRAGC ATG (SEQ ID NO: 370)5′-CTYGATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 371) S34D, E 5′-Phos-GTACAAAATCAAACCCTCCACAATGTCG BsrGI & SphITTGTTCTGATATGCGTTTAAATAGCTGTCATGANGC ATG (SEQ ID NO: 372)5′-CNTCATGACAGCTATTTAAACGCATATCAGAAC AACGACATTGTGGAGGGTTTGATTTT (SEQ IDNO: 373) A40V, D 5′-Phos-TCGACGCAGAAACATGTCCAACCGTAAA SphI & SalIGGTTATAGCAAWCACATGATTTGCATG (SEQ ID NO: 374)5′-CAAATCATGTGWTTGCTATAACCTTTACGGTTG GACATGTTTCTGCG (SEQ ID NO: 375)A40H, P 5′-Phos-TCGACGCAGAAACATGTCCAACCGTAAA SphI & SalIGGTTATAGCAAKGACATGATTTGCATG (SEQ ID NO: 376)5′-CAAATCATGTCMTTGCTATAACCTTTACGGTTG GACATGTTTCTGCG (SEQ ID NO: 377)A40Y, W 5′-Phos-TCGACGCAGAAACATGTCCAACCGTAAA SphI & SalIGGTTATAGCAMYAACATGATTTGCATG (SEQ ID NO: 378)5′-CAAATCATGTTRKTGCTATAACCTTTACGGTTG GACATGTTTCTGCG (SEQ ID NO: 379)F50D 5′-Phos-TCGACGCAATCACATGTCCAACCGTAAA SphI & SalIGGTTATAGCAAGCACATGATTTGCATG (SEQ ID NO: 380)5′-CAAATCATGTGCTTGCTATAACCTTTACGGTTG GACATGTGATTGCG (SEQ ID NO: 381)F50Q, P 5′-Phos-TCGACGCATKGACATGTCCAACCGTAAAG SphI & SalIGTTATAGCAAGCACATGATTTGCATG (SEQ ID NO: 382)5′-CAAATCATGTGCTTGCTATAACCTTTACGGTTG GACATGTCMATGCG (SEQ ID NO: 383)F50S, R 5′-Phos-TCGACGCAKCTACATGTCCAACCGTAAA SphI & SalIGGTTATAGCAAGCACATGATTTGCATG (SEQ ID NO: 384)5′-CAAATCATGTGCTTGCTATAACCTTTACGGTTG GACATGTAGMTGCG (SEQ ID NO: 385)V52D 5′-Phos-CAAATCATGTGCTTGCTATAACCTTTAC SphI & HindIIIGGGTGGACATGTTTCTGCGATGACATCACTGACTTCT GCTATGAACCATGTAAACCTTCTGAATAAA(SEQ ID NO: 386) 5′-AGCTTTTATTCAGAAGGTTTACATGGTTCATAGCAGAAGTCAGTGATGTCATCGCAGAAACATGTCCAA CCGTAAAGGTTATAGCAAGCACATGA TTTGCATG(SEQ ID NO: 387)

Secondary Screening of Individual Amino Acid Substitutions:Quadruplicate Testing in Microtiter Plates

For each chosen site, clones were selected corresponding to individualamino acid substitutions. These clones were grown, along with culturesof BBI-WT, BBIt-AV and BBIt-AV-F50T as controls, in 5 ml of Luria brothwith 5 ppm chloramphenicol (37° C., 250 rpm) in 15 ml culture tubes for˜10 hrs at 37° C. One hundred and fifty microliter aliquots of each thecultures were arrayed in the wells of a 96-well microtiter plate, 4wells for each amino acid substitution and each control. Each culturegrown in the arrayed plate was then used to inoculate duplicate filterplates (Millipore, MSGV2210) containing 150 μl of MBD medium with 5μg/ml chloramphenicol per well by using a pin replicator. The culturesin the plates were grown in the presence and absence of2-mercaptoethanol and analyzed as described above. BBIt-AV-F50T is amodified BBIt-AV that was identified as having an initial BBIt-AV:BCEactivity ratio prior to activation that was comparable to that of theunmodified BBIt-AV parent, but which has a three-fold greaterBBIt-AV:BCE activity ratio than that of the unmodified BBIt-AV afteractivation with a reducing agent such as 2-mercaptoethanol.

For each site, the BBI:BCE activity ratio was determined for each welland the average values for the quadruplicate measurements wascalculated. The activity ratios for the individual amino acidsubstitutions were compared to the ratios calculated for unmodifiedparent BBIt-AV (SEQ ID NO:187), the wild-type BBI (SEQ ID NO:13) and themodified BBIt-AV named BBIt-AV-F50T.

FIGS. 11A and 11B show the average of the quadruplicate determinationsof the BBI:BCE ratio determined in the presence of 2-mercaptoethanol(BME) relative to that determined for the sBBI for each of thesubstitutions at positions 50 and 37, respectively.

Overall, the data show single amino acid substitutions made at 16 of the66 positions/sites in the parent BBIt-AV molecule resulted in BBIt-AVvariants that had trypsin inhibitory activity equal or greater than thatof the precursor BBIt-AV (data summarized in Table 3). Of all the singlesite substitutions, F50R resulted in the best BBIt-AV:BCE− activityratio, while several substitutions at position 50 produced modifiedvariant BBIt-AVs having a four-fold greater BBIt-AV:BCE activity ratiosthan that of the precursor unmodified BBIt-AV, and that were comparableto that of the wild-type inhibitor (FIG. 11A-B).

These data show that a single substitution in the BBI-AV molecule canhave a significant effect on the activity of the BBI-AV.

TABLE 3 Substitutions that result in modified BBIt-AV molecules withgreater BBI:BCE activity ratios than the unmodified BBIt-AV PositionNative Amino Acid Improved Substitutions (with BME) 1 D A, P, 4 S V(4D13 insert)* 5 S P, A 11 Q G 13 A Y, I, F, M, L V, K, R 18 N I, V 25 SK N, W, I, A, R 27 M H, R, K, V, A, Q 29 L R, K, P 31 S Q, H, E, A, R,W, K, T 38 S N, K, R 40 A H, K, Q, R, Y 50 F R, Q, K, T, V, M, S 52 V K,T, R, Q, L, H, A, M, S, E 55 T M 65 S E D *The 4D13 insert is a 13 aminoacid sequence that was added to the N-terminus of the BBI protein asdescribed below.

Example 11 Selection of Modified Variants Having Improved PurificationYield

In this example, the methods described were used to identify modifiedBBIt-AVs containing single amino acid substitutions that retainedtrypsin inhibitory activity while being produced at yields greater thanthe corresponding unmodified precursor BBIt-AV.

Modified BBI-AVs that had been selected for having the greatestBBIt-AV:BCE activity ratio in the quadruplicate plate screens werefurther tested in a shake flask screen that was designed to mimic theacid/heat treatment that is used during the purification process tocleave the BBIt-AV from the BCE:BBIt-AV fusion protein. Thus, modifiedBBIt-AVs that retained a BBI:BCE activity ratio greater than that of theunmodified BBI-AV following acid/heat treatment were identified asmodified BBIt-AVs that would be produced at yields greater than that ofthe unmodified precursor BBIt-AV.

After activation with reducing agent, e.g. 2-mercaptoethanol, a higherBBIt-AV:BCE activity ratio indicates that a given amino acidsubstitution has significantly increased the fraction of molecules withat least a correctly folded trypsin inhibitory loop. However, twodifferent amino acid substitutions with similar BBIt-AV:BCE activityratios could have somewhat different total yields after purification.For example, V52L and M27A activate to similar levels but M27A has about40% better yield after acid/heat treatment. Thus, variants selected inthe first screens as “good activators” were further evaluated by asecond criterion that was developed to better predict purificationyield.

Shake Flask Screen: Testing for the Purification Yield

To increase expression, the gene copy of the selected clones wasamplified by sequentially streaking colonies on Luria agar plates with25 μg/ml chloramphenicol until the growth rate was similar to growth onLuria agar plates without chloramphenicol. Cultures of the selectedvariants and controls were grown in 3 ml Luria broth with 25 μg/mlchloramphenicol in 15 ml tubes for ˜10 h. These cultures (30 μl) werethen used to inoculate 30 ml of MBD medium in 250 ml baffled shakeflasks and grown at 37° C. and 225 rpm for ˜60 h.

For selection of the variants with the best yields, the culture brothswere processed as follows. The broth was first activated by mixing 20 mlof broth with 20 ml of 0.25 M glycine buffer (pH 9.3) and the pH wasadjusted to 9.0 with 50% NaOH. Then, 2-mercaptoethanol was added to thediluted cultures to a final concentration of 2 mM and the cultures wereincubated overnight at room temperature with gentle shaking. The BBIt-AVmoiety was then cleaved from the fusion protein by acid/heat treatment,which was accomplished by first adjusting the pH of the activated brothto pH 1.9-2.0 with sulfuric acid, and followed by incubation at 60° C.for 16 hours, with shaking. The BBIt-AV-BCE fusion protein and the BCEcatalytic domain are not soluble at pH 2, whereas the free BBI speciesis soluble. After the cleavage reaction, the insoluble material wasremoved by centrifugation (5 min. at 3,000 rpm) and the supernatant wasanalyzed for trypsin inhibitory activity as described above. The BBIactivity was also determined for the starting material and for thesamples taken after activation. The BCE activity of the startingmaterial was also determined as described above. The BBIt-AV:BCEactivity ratios were then calculated for each step in process. Themodified BBIt-AVs produced at the highest yields following the acid/heattreatment were selected for further study.

Amino acid substitutions were found at sites 13, 25, 27, 29, 31, 40, 50and 52 that significantly improved the BBIt-AV yield after acid/heattreatment. Other substitutions with improved yields after acid/heattreatment were D1C (when not activated), S4V, S5P, Q11G, S38N and S65E.An additional variant that was produced by an unexpected duplication ofa primer (during the QuikChange reaction used to make thesite-saturation library at position 4) also had a higher BBIt-AV:BCEactivity ratio after acid/heat treatment. In this variant, the aminoacid sequence, DDEPSKPCCDPDP (SEQ ID NO:389) (called the 4D13 insert),was inserted between the linker and the N-terminus of BBIt-AV (SEQ IDNO:187) to generate a modified BBIt-AV fused to the linker in SEQ IDNO:391.

(SEQ ID NO: 391) DNNDPIPDPddepskpccdpdpDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE.

The linker is shown in italicized letters, the insert is written inlower case letters and the BBIt-AV corresponding to SEQ ID NO:187 is inbold letters.

The sequence of the 4D13 modified BBtl-AV is:

(SEQ ID NO: 390) DDEPSKPCCDPDPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCFCVDITDFCYEPCKPSE.

The synthetic gene encoding 4D13-BBIt-AV of SEQ ID NO:390 is:

(SEQ ID NO: 394) GGATCCAGATGACGAACCGAGCAAACCTTGCTGTGATCCAGACCCTGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAAGCGAGTAAAAG CTT.

Example 12 Combinations of Mutations

In this example, the single amino acid substitutions that resulted inBBIt-AVs having improved BBI:BCE activity ratios in either the platescreens or in the shake flask screen were combined to generate modifiedBBIt-AVs that have greater BBIt-AV:BCE activity ratios and/or productionyields than those of modified BBIt-AVs carrying single amino acidsubstitutions.

Following the primary and secondary screens described in Examples 9 and10 above, three modified BBIt-AVs each containing a single amino acidsubstitution A13I, F50T and V52A, respectively, were identified ashaving an improved BBI:BCE activity ratio relative to the respectivecontrol. A library was constructed to select for the best combination ofthese three substitutions. The library was made by the QuikChange®mutagenesis protocol described above using equimolar concentrations ofthe following primers in the reaction mixture: primer5′-CTGTTGCGATCAATGCATTTGTACGAAATC (SEQ ID NO:395) was used to generatethe A13I substitution, primer5′-CTGTACGGGTGGACCTGTACATGCGYCGACATCACGGACTTC (SEQ ID NO:396) was usedto generate the F50T and F50T-V52A substitutions, and primer5′-GACCTGTTTTTGCGYCGACATCACGGAC (SEQ ID NO:397) was used to generate theV52A substitution in the reaction mixture. Clones were selected andscreened in microtiter plates and screened as described above.

The modified double variant BBIt-AV containing the F50T and the V52Asubstitutions (BBIt-AV-F50T-V52A; SEQ ID NO:595) was obtained, anddetermined to have the polynucleotide sequence of SEQ ID NO:398.

BBIt-AV-F50T-V52A (SEQ ID NO: 398)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 595) DPDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE.Generation of Modified BBIt-AVs Containing Three, Four and Five AminoAcid Substitutions

Combinations of four productive substitutions, A13I, S25L, L29P andA40K, identified in the screens described in Examples 9 and 10 abovewere made in the BBIt-AV-F50T-V52A double variant. These substitutionswere combined by using synthetic genes that were cloned into the BamHIand HindIII sites of the p2JMagk103Ink2BBIt-AV vector. Thepolynucleotide sequences encoding the corresponding amino acid sequencesfor the modified BBIt-AVs comprising combinations of substitutions aregiven below.

BBIt-AV-A13I-S25L-F50T-V52A (SEQ ID NO: 399)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 596) DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-A13I-S25L-L29P-F50T-V52A (SEQ ID NO: 400)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 597) DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE. BBIt-AV-A13I-S25L-L29P-A40K-F50T-V52A (SEQ ID NO:401) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 598) DPDDESSKPCCDQCICTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-A13I-S25L-A40K-F50T-V52A (SEQ ID NO: 402)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 599) DPDDESSKPCCDQCICTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-A13I-L29P-F50T-V52A (SEQ ID NO: 403)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 600) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACASCACYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-A13I-L29P-A40K-F50T-V52A (TA5) (SEQ ID NO:404) GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 601) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-A13I-A40K-F50T-V52A (SEQ ID NO: 405)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCATTTGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 602) DPDDESSKPCCDQCICTKSNPPQCRCSDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-S25L-F50T-V52A (SEQ ID NO: 406)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 603) DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-S25L-L29P-F50T-V52A (SEQ ID NO: 407)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 604) DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-S25L-L29P-A40K-F50T-V52A (SEQ ID NO: 408)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 605) DPDDESSKPCCDQCACTKSNPPQCRCLDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-S25L-A40K-F50T-V52A (SEQ ID NO: 409)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTCTTGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 606) DPDDESSKPCCDQCACTKSNPPQCRCLDMRLNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-L29P-F50T-V52A (SEQ ID NO: 410)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCGCATGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 607) DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCACYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-L29P-A40K-F50T-V52A (SEQ ID NO: 411)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCCGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 608) DPDDESSKPCCDQCACTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE BBIt-AV-A40K-F50T-V52A (SEQ ID NO: 412)GGATCCAGACGATGAGAGCTCTAAACCCTGTTGCGATCAATGCGCATGTACGAAATCAAATCCTCCACAGTGTCGGTGTTCCGATATGCGTCTGAATTCCTGTCATAGTGCCTGCAAAAGCTGCAAGTGTTATAACCTGTACGGGTGGACCTGTACATGCGCCGACATCACGGACTTCTGCTATGAACCATGTAAACCAA GCGAGTAAAAGCTT (SEQID NO: 609) DPDDESSKPCCDQCACTKSNPPQCRCSDMRNLSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSEGeneration of Modified BBIt-AVs Containing Six and Seven Amino AcidSubstitutions

Additional modified BBIt-AV were generated to comprise variationsincluding the 4D13 insertion, single amino acid substitutions D1C, S4V,S5P, Q11G, I13L, S25R, M27R, P29K, S31A, S31R, S38N, T50K, A52T, S65E,and double amino acid substitutions including S25R-S31R and S25R-S31with substitutions present in a quintuple variant(BBIt-AV-A13I-L29P-A40K-F50T-V52A) encoded by the polynucleotide of SEQID NO:404.

The 4D13 peptide was inserted into the p2JM103 based vector constructedfor the expression of BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:404)with an oligonucleotide cassette having BamHI and SacI restriction siteson the ends using the oligonucleotides:

(SEQ ID NO: 392) 5′-GATCCAGATGACGAACCGAGCAAACCTTGCTGTGATCCAGACCCTGACGATGAGAGCT and (SEQ ID NO: 393)5′-CTCATCGTCAGGGTCTGGATCACAGCAAGGTTTGCTCGGTTCGTCAT CTG.

Amino acid sequence of BBIt-AV-4D13 insert-A13I-L29P-A40K-F50T-V52A:

(SEQ ID NO: 413) DPDDEPSKPCCDPDPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCKCYNLYGWTCTCADITDFCYEPCKPSE

The other variants were made by cloning synthetic genes into the BamHIand HindIII sites of p2JM103-Ink2. The polynucleotide sequences of eachof the synthetic genes and resulting modified amino acid sequences ofthe modified variant BBIt-AVs are given in Table 4.

TABLE 4 BBIt-AVs comprising substitutions/modifications based on theBBIt-AV-A13I-L29P-A40K-F50T-V52A variant (SEQ ID NO: 601) Amino AcidAmino Acid Substitutions Synthetic Gene Sequence of the in BBIt-AVSequence Encoding the BBIt-AV Variant BBIt-AV Variant BBIt-AV-D1C-GGATCCATGCGATGAGAGCTCTAAACCTTGT DPCDESSKPCCDQ A13I-L29P-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52AATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 611)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 414) BBIt-AV-S4V-GGATCCAGACGATGAGGTTTCTAAACCTTGT DPDDEVSKPCCDQ A13I-L29P-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52AATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 612)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 415) BBIt-AV-S5P-GGATCCAGACGATGAGAGCCCTAAACCTTGT DPDDESPKPCCDQ A13I-L29P-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52AATAGCTGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 613)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 416) BBIt-AV-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDG Q11G-A13I-TGCGATGGCTGCATTTGTACTAAATCAAACC CICTKSNPPQCRCS L29P-A40K-CTCCACAATGTCGTTGTTCTGATATGCGTCCT DMRPNSCHSACKS F50T-V52AAATTCATGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCAATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSEGCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO: 614)ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 417) BBIt-AV-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ A13L-L29P-TGCGATCAATGCCTTTGTACTAAATCAAACCC CLCTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS V52AATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 615)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 418) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ S25R-L29P-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCR A40K-F50T-TCCACAATGTCGTTGTAGAGATATGCGTCCT DMRPNSCHSACKS V52AAATAGCTGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCAATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSEGCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO: 616)ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 419) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ S25R-L29P-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCR S31A-A40K-TCCACAATGTCGTTGTAGAGATATGCGTCCT DMRPNACHSACKS F50T-V52AAATGCTTGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCAATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSEGCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO: 617)ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 420) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ S25R-L29P-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCR S31R-A40K-TCCACAATGTCGTTGTAGAGATATGCGTCCT DMRPNRCHSACKS F50T-V52AAATCGCTGTCATTCTGCATGCAAATCATGTAA CKCYNLYGWTCTCAATGCTATAACCTTTACGGTTGGACATGTACAT DITDFCYEPCKPSEGCGCAGATATCACTGACTTCTGCTATGAACC (SEQ ID NO: 618)ATGTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 421) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ M27R-L29P-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATAGACGTCCTA DRRPNSCHSACKSC V52AATTCATGTCATTCTGCATGCAAATCATGTAAA KCYNLYGWTCTCADTGCTATAACCTTTACGGTTGGACATGTACATG ITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 619)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 422) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29K-A40K-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50T-V52ATCCACAATGTCGTTGTTCTGATATGCGTAAAA DMRKNSCHSACKSATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 620)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 423) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-S31A-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNACHSACKS V52AATGCTTGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 621)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 424) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-S31R-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNRCHSACKS V52AATAGATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 622)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 425) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-S38N-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS A40K-F50T-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKN V52AATAGCTGTCATTCTGCATGCAAAAACTGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 623)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 426) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-A40K-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50K-V52ATCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKSATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCKCTGCTATAACCTTTACGGTTGGACATGTAAATG ADITDFCYEPCKPSECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 624)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 427) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-A40K-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50T-V52TTCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKSATTCATGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCTTGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPSECACAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 625)GTAAACCTTCTGAATAAAAGCTT (SEQ ID NO: 428) BBIt-AV-A13I-GGATCCAGACGATGAGAGCTCTAAACCTTGT DPDDESSKPCCDQ L29P-A40K-TGCGATCAATGCATTTGTACTAAATCAAACCC CICTKSNPPQCRCS F50T-V52A-TCCACAATGTCGTTGTTCTGATATGCGTCCTA DMRPNSCHSACKS S65EATAGCTGTCATTCTGCATGCAAATCATGTAAA CKCYNLYGWTCTCATGCTATAACCTTTACGGTTGGACATGTACATG DITDFCYEPCKPEECGCAGATATCACTGACTTCTGCTATGAACCAT (SEQ ID NO: 626)GTAAACCTGAAGAATAAAAGCTT (SEQ ID NO: 429)

The BBI:BCE ratio of the modified variant BBIt-AVs was determined asdescribed above, and the yield for each of the modified variant BBIt-AVswas calculated. The modified BBIt-AVsBBIt-AV-A13I-S25R-L29P-S31R-A40K-F50T-V52A (SEQ ID NO:618),BBIt-AV-A13I-S25R-L29P-S31A-A40K-F50T-V52A (SEQ ID NO:617),BBIt-AV-A13I-S25R-L29P-A40K-F50T-V52A (SEQ ID NO:616),BBIt-AV-A13I-L29P-S31R-A40K-F50T-V52A (SEQ ID NO:622) had significantlybetter yields after acid/heat treatment than quintupleBBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601), whileBBIt-AV-A13I-L29P-A40K-F50K-V52A (SEQ ID NO:624),BBIt-AV-A13L-L29P-A40K-F50T-V52A (SEQ ID NO:615) and BBIt-AV-4D13insert-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:413) had slightly betteryields than BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:601). Themodified BBIt-AV-A13I-L29P-A40K-F50T-V52A-S65E (SEQ ID NO:626) andBBIt-AV-A13I-L29P-S38N-A40K-F50T-V52A (SEQ ID NO:623) had similar yieldsas BBIt-AV-A13I-L29P-A40K-F50T-V52 (SEQ ID NO:624),BBIt-AV-A13I-M27R-L29P-A40K-F50T-V52A (SEQ ID NO:619),BBIt-AV-A31I-L29P-S31A-A40K-F50T-V52A (SEQ ID NO:621) andBBIt-AV-A13I-L29P-A40K-F50T-V52T (SEQ ID NO:615) somewhat less, whileBBIt-AV-S4V-A31I-L29P-A40K-F50T-V52A (SEQ ID NO:612),BBIt-AV-Q11G-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:614),BBIt-AV-A13I-L29K-A40K-F50T-V52A (SEQ ID NO:620) andBBIt-AV-S5P-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:611) all hadsignificantly lower yields than BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ IDNO:601), after acid-heat treatment. The yield ofBBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:611) in the absence ofactivation was also much lower than BBIt-AV-A13I-L29P-A40K-F50T-V52A(SEQ ID NO:601). However, the yield after acid/heat treatment ofBBIt-AV-D1C-A13I-L29P-A40K-F50T-V52A (SEQ ID NO:611) when not activatedwas about two fold higher than BBIt-AV-A13I-L29P-A40K-F50T-V52A (SEQ IDNO:601) when activated but similar to BBIt-AV-A13I-L29P-A40K-F50T-V52A(SEQ ID NO:601) when it was not activated. Thus, no substitutions werefound that could produce high yields without activation.

Generation of Modified BBI-AVs Containing Eight Amino AcidSubstitutions: Octuple Variants

Following the testing of the quintuple-based modified BBI-AVs,additional modified variant BBIs (octuple variants) were generated tocontain eight amino acid substitutions. The activity and purificationyield of the octuple variants were determined as described above.Construction of the octuple modified BBIt-AVs was achieved as describedabove using the synthetic genes shown in Table 5.

TABLE 5 Octuple Modified Variant BBIt-AVs Amino Acid Synthetic GeneSequence Amino Acid Sequence Substitutions Encoding the Octuple Variantof the BBIt-AV Variant BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTTDPDDESSKPCCDQCICTKSNP S25R-M27A- GTTGCGATCAATGCATCTGTACAAAATCAPQCRCRDARPNACHSACKSC L29P-S31A- AACCCTCCACAATGTCGTTGTAGAGATGCHCYNLYGWTCKCTDITDFCYE A40H-F50K- TCGTCCTAATGCATGTCATTCTGCATGCA PCKPSEV52T (KT8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO: 627)GGACATGTAAATGCACAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT(SEQ ID NO: 486) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTTDPDDESSKPCCDQCICTKSNP S25K-M27A- GTTGCGATCAATGCATCTGTACAAAATCAPQCRCKDARRNECHSACKSC L29R-S31E- AACCCTCCACAATGTCGTTGTAAAGATGCKCYNLYGWTCQCQDITDFCYE A40K-F50Q- TCGTAGAAATGAATGTCATTCTGCATGCA PCKPSEV52Q (QQ8) AATCATGTAAATGCTATAACCTTTACGGTT (SEQ ID NO: 628)GGACATGTCAATGCCAAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT(SEQ ID NO: 487) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTTDPDDESSKPCCDQCICTKSNP S25K-M27R- GTTGCGATCAATGCATCTGTACAAAATCAPQCRCKDRRENACHSACKSC L29E-S31A- AACCCTCCACAATGTCGTTGTAAAGATAGHCYNLYGWTCRCKDITDFCYE A40H-F50R- ACGTGAAAATGCTTGTCATTCTGCATGCA PCKPSEV52K (RK8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO: 629)GGACATGTAGATGCAAAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT(SEQ ID NO: 488) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTTDPDDESSKPCCDQCICTKSNP S25K-M27A- GTTGCGATCAATGCATCTGTACAAAATCAPQCRCKDARRNACHSACKSC L29R-S31A- AACCCTCCACAATGTCGTTGTAAAGATGCHCYNLYGWTCRCLDITDFCYE A40H-F50R- TCGTAGAAATGCTTGTCATTCTGCATGCA PCKPSEV52L (RL8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO: 630)GGACATGTAGATGCTTAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT(SEQ ID NO: 489) BBIt-AV-A131- GGATCCAGACGATGAGAGCTCTAAACCTTDPDDESSKPCCDQCICTKSNP S25K-M27Q- GTTGCGATCAATGCATCTGTACAAAATCAPQCRCKDQRPNECHSACKSC L29P-F50R- AACCCTCCACAATGTCGTTGTAAAGATCAHCYNLYGWTCRCQDITDFCYE A40H-F50R- ACGTCCTAATGAATGTCATTCTGCATGCA PCKPSEV52Q (RQ8) AATCATGTCACTGCTATAACCTTTACGGTT (SEQ ID NO: 631)GGACATGTAGATGCCAAGACATCACTGAC TTCTGCTATGAACCATGTAAACCTTCTGAA TAAAAGCTT(SEQ ID NO: 490)

The best octuple variants selected in the shake flask screen were testedin the purification process essentially as described above, andincluding the following changes: 1) after growth, the culture was twofold diluted in glycine buffer (125 mM final concentration and pHadjusted to 9.0) instead of CHES buffer; 2) samples were activated with2 mM 2-mercaptoethanol only (no sodium sulfite was added; 3) the acidprecipitated fusion protein was resuspended in 100 ml of 125 mM glycine(rather than 350 ml of 40 mM glycine); and 4) the pellet collected afterthe acid/heat treatment was washed with 10 ml water, filtered, and this“washed pellet” filtrate was combined with the original filtrate. Thefinal filtrates were concentrated and trimmed with Glu-BL as describedabove

The results show that octuple variant BBIt-AV molecules QQ8 (SEQ IDNO:628) and KT8 (SEQ ID NO:627) resulted in purification yields thatwere greater than that of the quintuple modified variantBBIt-AV-A13I-L29P-A40K-F50T-V52A variant (SEQ ID NO:601). The modifiedoctuple variants BBIt-AV-A13I-S25K-M27A-L29R-S31A-A40H-F50R-V52L (RL8;SEQ ID NO:630), BBIt-AV-A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q (QQ8;SEQ ID NO:628) and BBIt-AV-A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T (KT8;SEQ ID NO:627) resulted in yields that were better than the wild-typesBBI molecule/protein (FIG. 13).

Example 13 Improved Production of BBI Scaffolds Carrying FGF5 or TGFβ1Binding Peptides

In this example, the same amino acid substitutions that resulted inmodified variant BBIt-AVs having improved trypsin inhibitory activityand/or purification yields (see Examples 7-13) were tested for improvingthe trypsin inhibitory activity and/or the purification yield of variantBBI scaffolds in which the chymotrypsin loop was replaced with either anFGF-5 or a TGFβ1 binding peptide.

Generation of Variant and Modified Variant BBI-FGF-5 Proteins:

Modified variant BBIt-FGF-5 protease inhibitors comprising thecombination of substitutions A13I-L29P-F50T-V52A (all with alanine atposition 40) and either the FGF-5 binding peptide MM007: RTQPYPL (SEQ IDNO:670), or FGF-5 binding peptide FGFps2: TWIDSTP (SEQ ID NO:671) inplace of the chymotrypsin loop, were constructed by ligating a syntheticgene into the BamHI and HindIII sites of the vectorp2JMagk103-Ink2-BBIt-AV. The amino acid sequences and DNA sequences ofthe synthetic genes encoding the resulting modified variantMM007-Q-BBIt-FGF-5 (SEQ ID NO:432; SEQ ID NO:433) and the modifiedvariant FGFps2-Q-BBIt-FGF-5, respectively (SEQ ID NO:434 SEQ ID NO:435)are as follows.

MM007-Q-BBIt-FGF-5 synthetic gene (SEQ ID NO: 433)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTACTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGCTGTCATTCTGCATGCAAATCATGTGCTTGCCGTACTCAACCATACCCTCTTTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTTMM007-Q-BBIt-FGF-5 protein (SEQ ID NO: 432)DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACRTQPYPL CTCADITDFCYEPCKPSEFGFps2-Q-BBIt-FGF-5 (SEQ ID NO: 435)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTACTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGCTGTCATTCTGCATGCAAATCATGTGCTTGCACTTGGATTGATTCAACACCATGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTT (SEQID NO: 434) DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACTWIDSTPCTCADITDFCYEPCKPSEGeneration of Variant and Modified Variant BBI-TGFβ Proteins:

The chymotrypsin inhibitory loop of the BBIt-AV of SEQ ID NO:187 wasreplaced with the TGFβ1 binding peptide PEN3: CLCPENINVLPCN (SEQ IDNO:436), the TGFβ1 binding peptide MM021W: CICKHNVDWLCF (SEQ ID NO:437)or the TGFβ1 binding peptide WTQ: CICWTQHIHNCF (SEQ ID NO:438) togenerate variant BBPIs according to the method described in Example 2,as follows.

Oligonucleotide pairs were used to make cassettes to replace the FGFhIbinding peptide in the chymotrypsin reactive site loop encoded by thep2JM103-Ink2-2BBI-FGFhI expression vector with the TGFβ1 bindingpeptides, PEN3, MM021W or WTQ. The p2JM103-Ink2-2BBI-FGFhI wasconstructed using the primers (SEQ ID NO:91 and SEQ ID NO:92) andQuikChange® method as described in Example 2. The TGFβ binding cassetteswere ligated into the SphI and SalI restriction sites in the vectorp2JM103Ink2-2BBI-FGFhI to construct the variant BBI moleculesPEN3-BBIt-TGFβ₁, MM021W-BBIt-TGFβ and WTQ-BBIt-TGFβ₁, respectively. TheDNA sequences of the oligonucleotides used to make the cassettes areshown below.

PEN3 (2^(nd) loop) (SEQ ID NO: 439)CAAAAGCTGTCTTTGTCCTGAAAATATTAACGTTCTTCCTTGTAACTGCG And (SEQ ID NO: 440)TCGACGCAGTTACAAGGAAGAACGTTAATATTTTCAGGACAAAGACAGCT TTTGCATG MM021W(2^(nd) loop) (SEQ ID NO: 129)CAAATCATGCATTTGTAAACACAACGTAGATTGGTTATGTTTTTGCG And (SEQ ID NO: 130)TCGACGCAAAAACATAACCAATCTACGTTGTGTTTACAAATGCATGATTT GCATG WTQ (2^(nd)loop) (SEQ ID NO: 441) CAAATCATGCATTTGTTGGACACAACATATCCACAACTGTTTTTGCGAnd (SEQ ID NO: 442) TCGACGCAAAAACAGTTGTGGATATGTTGTGTCCAACAAATGCATGATTTGCATG

In addition, modified variant BBPI-TGFβ1 variants comprising thecombination of substitutions A131-L29P-V52A (all also have alanine atposition 40 and threonine at position 50) and the TGFβ1 binding peptidePEN3-Q: CPENINVLPC (SEQ ID NO:672), MMO21W-Q: CKHNVDWLC (SEQ ID NO:673)or WTQ-Q: CWTQHIHNC (SEQ ID NO:674) in place of the chymotrypsin loop,were constructed by ligating a synthetic gene into the BamHI and HindIIIsites of the vector p2JMagk103-Ink2-BBIt-AV. The amino acid sequencesand DNA sequences of the synthetic genes encoding the resulting modifiedvariant PEN3-Q -BBIt-TGFβ1 (SEQ ID NO:443; SEQ ID NO:444), the modifiedvariant MMO21W-BBI-TGFβ1 (SEQ ID NO:445; SEQ ID NO:446), and themodified variant WTQ-BBI-TGFβ1 (SEQ ID NO:447; SEQ ID NO:448) are asfollows.

PEN3-Q-BBIt-TGFβ1 (SEQ ID NO: 443)DPDDESSKPCCDQCICTKSNPPCQCRCSDMRPNSCHSACKSCACPENINV LPCTCADITDFCYEPCKPSEPEN3-Q-BBIt-TGFβ1 (SEQ ID NO: 444)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTACTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGCTGTCATTCTGCATGCAAATCATGTGCTTGCCCAGAAAACATCAACGTTCTTCCTTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAAC CATCTGAATAAAAGCTTMM021W-Q-BBIt-TGFβ1 (SEQ ID NO: 445)DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACKHNVDWL CTCADITDFCYEPCKPSEMM021W-Q-BBIt-TGFβ1 (SEQ ID NO: 446)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTACTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGCTGTCATTCTGCATGCAAATCATGTGCTTGCAAACATAACGTTGATTGGCTTTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTTWTQ-Q-BBIt-TGFβ1 (SEQ ID NO: 447)DPDDESSKPCCDQCICTKSNPPQCRCSDMRPNSCHSACKSCACWTQHIHN CTCADITDFCYEPCKPSEWTQ-Q-BBIt-TGFβ1 (SEQ ID NO: 448)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATTTGTACTAAATCAAATCCTCCACAATGTCGTTGTTCTGATATGCGTCCTAATAGCTGTCATTCTGCATGCAAATCATGTGCTTGCTGGACACAACATATCCACAACTGTACATGCGCAGACATCACTGACTTCTGCTATGAACCATGTAAACCAT CTGAATAAAAGCTT

The ten vectors comprising the variant and modified variant BBIscontaining an FGF-5 or a TGFβ binding peptide were used to transform B.subtilis BG6006 host cells. The transformants were grown and theengineered BBIs produced by the host cells were tested for trypsininhibitory activities and production yields as described above.

FIG. 14 shows the trypsin inhibitory activity of the unmodified variantparent MM007-PT-BBIt-FGF-5 (SEQ ID NO:678):(DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC RTQPYPL CFCVDITDFCYEPCKPSE;SEQ ID NO:678), the unmodified variant parent FGFps2-PT-BBIt-FGF5 (SEQID NO:679): (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC TWIDSTPCFCVDITDFCYEPCKPSE; SEQ ID NO:679), the unmodified variant parentPEN3-PT-BBIt-TGFβ₁ (SEQ ID NO:680):(DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC PENINVLP CFCVDITDFCYEPCKPSE;SEQ ID NO:680), the unmodified variant parent WTQ-PT-TGFβ₁ (SEQ IDNO:681): (DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC WTQHIHNCFCFCVDITDFCYEPCKPSE; SEQ ID NO:681), and the unmodified variant parentMM0021W-PT-TGFβ₁ (SEQ ID NO:682):(DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCAC KHNVDWL CFCVDITDFCYEPCKPSE;SEQ ID NO:682), (designated PT in FIG. 14) and the correspondingmodified variant MM007-Q-BBIt-FGF-5 (SEQ ID NO:432), the modifiedvariant FGFps2-Q-BBIt-FGF5 (SEQ ID NO:434), the modified variantPEN3-Q-BBIt-TGFβ1 (SEQ ID NO:443), the modified variant WTQ-Q-BBIt-TGFβ1(SEQ ID NO:445), and the modified variant MM0021W-Q-BBIt-TGFβ1 (SEQ IDNO:447) proteins (designated Q in FIG. 14) measured after growth, afteractivation with 2-mercaptoethanol and after acid/heat treatment. Thedata show that the combination of the amino acids 13I-29P-40A-50T-52Awhen present in the BBIt scaffold carrying FGF-5 or TGFβ1 bindingpeptides improve the trypsin inhibitory activity after growth andactivation, and the production yield after acid/heat treatment whencompared to the corresponding parent BBI scaffold carrying the bindingpeptides.

The data also evidences that substitutions that were shown to improvethe trypsin inhibitory activity and/or purification yield of a modifiedvariant BBI in which the chymotrypsin loop had been replaced with a VEGFbinding peptide are not specific to BBI-AVs as the same substitutionsimprove the trypsin inhibitory activity and/or the production yield ofBBIs in which the chymotrypsin loop was replaced by other bindingpeptides e.g. FGF-5 binding peptides and TGFβ1 binding peptides.

Therefore, the amino acid substitutions made in the BBI scaffold thatincrease the production yield and/or the trypsin activity of BBIt-AV areexpected to be generally applicable to BBI scaffolds carrying otherbinding peptides.

Example 14 Performance of Bowman Birk Inhibitor Scaffolds

In this example, the ability to predict the effect of amino acidsubstitutions on the trypsin activity and/or production yield of avariant Bowman Birk Inhibitor e.g. BBIt-AV (SEQ ID NO:187) was tested bysubstituting amino acids that are not conserved across Bowman BirkInhibitor sequences from Dolichos biflorus (BBdb, Acc. No. AAK97765; SEQID NO:449)PSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCVCTFSIPAQCVCVDMKDFCYEPCK (SEQ IDNO:449), from Glycine max (soybean) protease inhibitor IV or D-II(BBsb3, Acc. No. P01064; SEQ ID NO: 450)DDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCMCTRSQPGQCRCLDT NDFCYKPCKSRDD(SEQ ID NO:450), and from Torresea (Amburana) cearensis (BBtc, Acc. No.P83283; SEQ ID NO:451)SSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACTHSIPAQCRCFDITDFCYKPCSG (SEQ IDNO:451), into the BBIt-AV scaffold.

Variant Bowman Birk Inhibitors were generated by replacing the secondprotease inhibitory loop of the Bowman Birk Inhibitor from Dolichosbiflorus (BBdb, Acc. No. AAK97765; SEQ ID NO:449), the second proteaseinhibitory loop of the Bowman Birk Inhibitor from Glycine max (soybean)protease inhibitor IV or D-II (BBsb3, Acc. No. P01064; SEQ ID NO: 450)and the second protease inhibitory loop of the Bowman Birk Inhibitorfrom Torresea (Amburana) cearensis (BBtc, Acc. No. P83283; SEQ IDNO:451) with the VEGF binding peptide CK37281 (ACYNLYGWTC; SEQ ID NO:9)to generate the corresponding variant BBdb-AV (SEQ ID NO:452), BBsb3-AV(SEQ ID NO:453), and BBtc-AV (SEQ ID NO:454).

The sequences were aligned, as shown in FIG. 15, and amino acids atpositions equivalent to positions 11, 13, 18, 23, 25, 27, 35, 37, 52, 54and 55 of the BBPI of SEQ ID NO:187 (BBIt-AV) were identified as notbeing conserved across the four inhibitors, and were chosen to modifythe BBIt-AV scaffold.

The synthetic genes encoding BBdb-AV (SEQ ID NO:455) and BBsb3-AV (SEQID NO:456) were ligated into the p2JMagk103-Ink2-BBIt-AV asBamHI-HindIII fragments. For cloning the BBtc-AV gene, the gene wasfirst digested with BfrI, the overhang filled in with T4 DNA polymeraseand dNTP's, the DNA purified, and then digested with BamHI. Thisfragment was ligated into the p2JMagk103-Ink2-BBIt-AV vector that hadbeen digested with HindIII, the overhang filled in with T4 DNApolymerase and dNTP's, the DNA purified, and then digested with BamHI.The DNA sequences and the corresponding amino acid sequences of thevariant PIs are as follows.

BBdb-AV (SEQ ID NO: 455)GGATCCTTCTGAGAGCTCTAAACCATGCTGTGATCAATGCGCTTGTACAAAATCTATCCCTCCACAATGCCGTTGCACTGATGTTCGTCTTAACTCATGTCACTCTGCATGCAGCTCATGCGCTTGTTACAACCTTTACGGTTGGACATGCGTTTGCGTCGACATGAAAGATTTCTGCTACGAACCTTGTAAATAAAAGC TT BBdb-AV (SEQ IDNO: 452) DPSESSKPCCDQCACTKSIPPQCRCTDVRLNSCHSACSSCACYNLYGWTCVCVDMKDFCYEPCK BBsb3-AV (SEQ ID NO: 456)GGATCCAGATGACGAATACTCTAAACCTTGCTGTGATCTTTGCATGTGTACACGTTCTATGCCACCTCAATGCTCATGTGAAGACATCCGCCTTAACTCTTGCCACTCAGATTGCAAAAGCTGCGCTTGTTACAACCTTTACGGTTGGACATGCCGTTGTTTAGATACTAACGATTTCTGCTACAAACCTTGCAAATCTC GTGATGATTAAAAGCTTBBsb3-AV AA (SEQ ID NO: 453)DPDDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCACYNLYGWT CRCLDTNDFCYKPCKSRDDBBtc-AV (SEQ ID NO: 457)GGATCCTTCTTCAAAATGGGAAGCTTGCTGTGATCGTTGCGCATGCACAAAATCTATCCCTCCACAATGCCACTGCGCTGATATCCGTCTTAACTCATGCCATTCTGCATGCGAAAGCTGCGCTTGTTACAACCTTTACGGTTGGACATGCCGTTGCTTCGATATCACTGATTTCTGTTACAAACCTTGCTCTGGCTAAAAGCTTAAAAGGAGACCGTTAATCTAAAATCATTATTTGAGGCCCGAGCTT AAAGCTTAAG BBtc-AV AA(SEQ ID NO: 454) DPSSKWEACCDRCACTKSIPPQCHCADIRLNSCHSACESCACYNLYGWTCRCFDITDFCYKPCSG

The three expression vectors comprising the variant inhibitor sequenceswere used to transform B. subtilis host cells BG6006.

Typically, substitutions at the N-terminal and C-terminal ends, whichare outside of the first disulfide bond (C8-C62), have small effects onthe trypsin inhibitory activity and/or purification yield. Thus, onlysubstitutions of amino acids at positions between C8 and C62 werestudied. The substitutions targeted in this study are shown in FIG. 15(bold and underlined).

The individual amino acid substitutions at positions 11, 13, 18, 23, 25,27, 35, 37, 50, 52, 54, 55, and 60 in BBIt-AV (SEQ ID NO:187) wereconstructed as described in Examples 8 and 10. These substitutions wereanalyzed by screening in microtiter plates (with and without reducingagent) as described in Examples 9 and 10. The results are summarized inTable 6 below

TABLE 6 Effect of single amino acid substitutions present in theBBtc-AV, BBdb-AV and BBsb3-AV scaffolds on the trypsin activity of themodified variant BBIt-AV Amino acid present in the alternative scaffoldand relative activity of the amino acid substitution Position and aminoBBItc-AV BBIdb-AV BBIsb3-AV acid present in the (SEQ ID (SEQ ID (SEQ IDBBIt-AV scaffold NO: 454) NO: 452) NO: 453) Q11 R (+/−) wt* L (+/−)**A13 wt wt M (+)*** N18 I (+++) I (+++) M (+/−) R23 H (−) wt S (−−−) S25A (+) T (+/−) E (−−−) M27 I (+/−) V (+/−) I (+/−) A35 wt wt D (+/−) K37E (+/−) S (+/−) wt F50 R (++) V (+) R (++) V52 F (+/−) wt L (+) I54 wt M(−) T (−)**** T55 wt K (−) N (+/−) E60 K (+/−) wt K (+/−) *wt indicatesthat the amino acid at this position is the same as in the BBIt-AVscaffold (SEQ ID NO: 187) **(+/−) indicates that the relative BBI:BCEactivity ratio in the presence of reducing agent is not significantlydifferent than that of BBIt-AV. ***(+) indicates that the the relativeBBI:BCE activity ratio in the presence of reducing agent is greater thanthat of BBIt-AV; the greater the number of “+”, the greater thedifference. ****(−)indicates that the the relative BBI:BCE activityratio in the presence of reducing agent is less than that of BBIt-AV;the greater the number of “−”, the greater the difference.

Assuming the effects of the amino acid substitutions are additive, thedata provided in Table 6 predicts that the variant BBItc-AV wouldperform significantly better than BBIt-AV, while BBIsb3-AV, shouldperform significantly worse than BBIt-AV. In addition, one would predictthat BBIdb-AV would perform somewhat better than BBIt-AV in terms ofactivation with reducing agent.

To test these predictions, the trypsin activity and production yield ofBBIdb-AV, BBIsb3-AV and BBItc-AV were tested using the Shake FlaskScreen described in Example 11 and their activity compared to that ofthe unmodified variant BBIt-AV.

The data shown in FIG. 16 show that, as predicted, BBItc-AV had thehighest trypsin inhibitory activity after activation, and BBIdb-AVinhibitory activity was also greater than that of the BBIt-AV inhibitorbut less than that of the BBItc-AV inhibitor. In addition, and aspredicted, BBIsb3-AV had very low trypsin inhibitory activity and didnot activate with reducing agent (inhibitory activity was too small tobe shown in FIG. 16). Furthermore, not only did BBItc-AV and BBIdb-AVhave higher inhibitory activities after activation than BBIt-AV, theyalso had higher inhibitory activities after acid/heat treatmentindicating improved production yields.

Therefore, the results indicate that activity data obtained when testingsingle amino acid substitutions in BBIt-AV can be used to predict theperformance of other Bowman Birk Inhibitors when used as scaffolds withbinding peptides replacing the second protease inhibitory loop

Example 15 Binding of Modified Variant BBIs to Target Proteins

In this example, the capacity of variant peptides to retain theirability to bind the corresponding target proteins was tested when thevariant peptides were grafted into a modified Bowman Birk Inhibitorscaffold to replace the chymotrypsin loop.

The construction of the expression vector for BBI is described inExample 1 and the constructions of vectors for variant BBIs containingthe VEGF-binding peptide CK37281 (SEQ ID NO:9), the FGF-5-bindingpeptide MM007 (SEQ ID NO:430) or the FGF-5-binding peptide FGF5ps2 (SEQID NO:431) in place of the chymotrypsin inhibitory sequence aredescribed in Example 13 above. The method for the construction ofexpression vectors encoding for the modified variant BBIt-AVs havingvarious combinations of amino acid substitution is described in Example12 and the method for constructing the FGF5 and TGFβ-binding BBIs isdescribed in Example 13.

VEGF-binding peptides named VegK (KYYLYWW; SEQ ID NO:458), VegT(TLWKSYW; SEQ ID NO:459) and VegKD (KYDLYWW; SEQ ID NO: 460) wereintroduced into the chymotrypsin inhibitory loop by ligatingoligonucleotide cassettes into the SphI and SalI sites ofp2JM103-Ink2-2BBIck81. The sequences of the oligonucleotides used togenerate the modified variant VEGK and VEGT BBPIs are shown below.

VegK: (SEQ ID NO: 461) CAAATCTTGCGCATGTAAATATTACCTTTACTGGTGGTGTTTTTGCGAnd (SEQ ID NO: 462) TCGACGCAAAAACACCACCAGTAAAGGTAATATTTACATGCGCAAGATTTGCATG BBIT-VEGK (SEQ ID NO: 640)DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYYLYWW CKCTDITFDCYEPCKPSEVegT: (SEQ ID NO: 463) CAAATCTTGCGCGTGCACACTTTGGAAATCTTACTGGTGTTTTTGCGAnd (SEQ ID NO: 464) TCGACGCAAAAACACCAGTAAGATTTCCAAAGTGTGCACGCGCAAGATTTGCATG BBIt-VEGT (SEQ ID NO: 641)DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACTLWKSYW CKCTDITDFCYEPCKPSEVegKD: (SEQ ID NO: 465) CAAATCTTGCATCTGTAAATATGATCTTTACTGGTGGTGTTTTTGCGAnd (SEQ ID NO: 466) TCGACGCAAAAACACCACCAGTAAAGATCATATTTACAGATGCAAGATTTGCATG

The VEGF-binding peptide named VegKD was introduced into the variantBBI-A13I-S25K-L29P-V52K by ligating a synthetic gene (VegKD-Q) into theBamHI and HindIII sites of p2JMagk103-Ink2-2BBIck81. The sequence of thesynthetic gene is shown and encoded BBPI are shown below.

VegKD-Q: (SEQ ID NO: 467)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAAAGATATGCGTCCTAATAGCTGTCATTCTGCATGCAAATCATGTATCTGCAAATATGACCTTTACTGGTGGTGTTTCTGCAAAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 642) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKYDLYWWCKCTDITDFCYEPCKPSE

The VEGF-binding peptides V1 (SKHSQIT; SEQ ID NO:468), V2 (KTNPSGS; SEQID NO:469), V3 (RPTGHSL; SEQ ID NO:470), V4 (KHSAKAE; SEQ ID NO:471), V5(KPSSASS; SEQ ID NO:472) and V6 (PVTKRVH; SEQ ID NO:473), and theTNFα-binding peptides T1 (RYWQDIP; SEQ ID NO:474), T2 (APEPILA; SEQ IDNO:475) and T3 (DMIMVSI; SEQ ID NO:476), were introduced into thechymotrypsin inhibitory loop of a modified BBIt containing amino acidsubstitutions A131-S25R-M27A-L29P-S31A-I40A-F50K-V52T by ligatingsynthetic genes into the BamHI and HindIII sites ofp2JMagk103-Ink2-2BBIck81. The DNA sequences of the synthetic genesencoding the resulting modified BBIs are shown below.

BBIt-AV-V1: (SEQ ID NO: 477)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCAGCAAACACTCTCAAATTACTTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 491) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACSKHSQITCKCTDITDFCYEPCKPSE BBIt-AV-V2: (SEQ ID NO: 478)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCAAAACAAACCCAAGCGGTTCTTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 632) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKTNPSGSCKCTDITDFCYEPCKPSE BBIt-AV-V3: (SEQ ID NO: 479)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCAGACCAACTGGTCACAGCCTTTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 633) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACRPTGHSLCKCTDITDFCYEPCKPSE BBIt-AV-V4: (SEQ ID NO: 480)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCAAACACAGCGCTAAAGCAGAATGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 634) DPDDESSKPCCDQCICTKSNPPQCRCRCARPNACHSACKSCACKHSAKAECKCTDITDFCYEPCKPSE BBIt-AV-V5: (SEQ ID NO: 481)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCAAACCAAGCTCTGCTTCATCTTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 635) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACKPSSASSCKCTDITDFCYEPCKPSE BBIt-AV-V6: (SEQ ID NO: 482)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCCCAGTTACTAAAAGAGTACACTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 636) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACPVTKRVHCKCTDITDFCYEPCKPSE BBIt-TNF-T1: (SEQ ID NO: 483)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCAGATACTGGCAAGATATTCCATGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 637) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACRYWQDIPCKCTDITDFCYEPCKPSE BBIt-TNF-T2: (SEQ ID NO: 484)GGATCCAGACGATGAGAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCGCACCAGAACCTATTCTTGCTTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 638) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACAPEPILACKCTDITDFCYEPCKPSE BBIt-TNF-T3: (SEQ ID NO: 485)GGATCCAGACGATGACAGCTCTAAACCTTGTTGCGATCAATGCATCTGTACAAAATCAAACCCTCCACAATGTCGTTGTAGAGATGCTCGTCCTAATGCATGTCATTCTGCATGCAAATCATGTGCTTGCGATATGATTATGGTTAGCATCTGTAAATGCACAGACATCACTGACTTCTGCTATGAACCATGTAAACCTT CTGAATAAAAGCTT (SEQID NO: 639) DPDDESSKPCCDQCICTKSNPPQCRCRDARPNACHSACKSCACDMIMVSICKCTDITDFCYEPCKPSE

The resulting vectors were used to transform B. subtilis BG6006 hostcells. Cultures were grown in 500 ml of MBD medium and the BBI specieswere purified essentially as described above and by Vogtentanz et al.,2007 (Protein Expr Purif 55:40-52 [2007]). BioVeris binding assays wereperformed essentially as outlined in Example 5 and by the manufacturer.Binding of the modified variant BBIs to their respective target proteinswas measured using a BioVeris® binding assay, which is a competitionassay that determines the binding of the modified variant BBIs to itstarget protein (e.g. VEGF, FGF5, TGFβ and TNFα) in the presence orabsence of a labeled competitor. The competitor can be a labeledmonoclonal antibody raised against the target protein or a labelednative receptor of the target protein. The electrochemiluminesentbinding assays were performed essentially as described in Example 5. Theapparent IC50s were determined from binding data by determining theconcentration of the BBI species needed to reduce the BioVeris signal byone half. BBI scaffolds without the target binding peptide in thechymotrypsin inhibitory loop were used as negative controls.

The results are summarized in Table 7 below.

TABLE 7 Binding activity of modified variant Bowman Birk Inhibitors inwhich the chymotrypsin loop was replaced by a VEGF-, FDF-5-, TGFβ-, orTNFα-binding variant peptide Variant Variant Resulting Modified TargetPeptide Peptide Bowman Birk Inhibitor Variant Bowman BioVeris ProteinName^(•) Sequence^(▴) Scaffold Birk Inhibitor Binding* VEGF CK37281YNLYGWT BBIt BBIt-AV of +++ (SEQ ID NO: 676) SEQ ID NO: 187 VEGF CK37281YNLYGWT BBIt-A13I-A40K-F50T-V52A BBIt-AV of +++ (SEQ ID NO: 676) SEQ IDNO: 602 VEGF CK37281 YNLYGWT BBIt-A13I-L29P-A40K-F50T- BBIt-AV of +++(SEQ ID NO: 676) V52A SEQ ID NO: 601 VEGF CK37281 YNLYGWTBBIt-A13I-S25R-M27A-L29P- BBIt-AV of +++ (SEQ ID NO: 676)S31A-A40H-F50K-V52T SEQ ID NO: 627 (KT8) VEGF CK37281 YNLYGWTBBIt-A13I-S25K-M27A-L29R- BBIt-AV of +++ (SEQ ID NO: 676)S31E-A40K-F50Q-V52Q SEQ ID NO: 628 (QQ8) VEGF CK37281 YNLYGWTBBIt-A13I-S25K-M27A-L29R- BBIt-AV of +++ (SEQ ID NO: 676)S31A-A40H-F50R-V52L SEQ ID NO: 630 (RL8) VEGF CK37281 YNLYGWT BBdbBBdb-AV +++ (SEQ ID NO: 676) SEQ ID NO: 452 VEGF CK37281 YNLYGWT BBtcBBtc-AV +++ (SEQ ID NO: 676) SEQ ID NO: 454 VEGF Veg K KYYLYWW BBItBBIt-AV of +++ (SEQ ID NO: 458) SEQ ID NO: 640 VEGF Veg T TLWKSYW BBItBBIt-AV of +++ (SEQ ID NO: 459) SEQ ID NO: 641 VEGF Veg KD KYDLYWW BBItBBIt-AV of +++ (SEQ ID NO: 460) SEQ ID NO: 462 VEGF Veg KD KYDLYWWBBIt-A13I-S25K-L29P-V52K BBIt-AV of +++ (SEQ ID NO: 460) SEQ ID NO: 643VEGF V1 SKHSQIT BBIt-A13I-S25R-M27A-L29P- BBIt-AV- ++ (SEQ ID NO: 468)S31A-F50K-V52T V1 of SEQ ID NO: 491 VEGF V2 KTNPSGSBBIt-A13I-S25R-M27A-L29P- BBIt-AV- +++ (SEQ ID NO: 469) S31A-F50K-V52TV2 of SEQ ID NO: 632 VEGF V3 RPTGHSL BBIt-A13I-S25R-M27A-L29P- BBIt-AV-++ (SEQ ID NO: 470) S31A-F50K-V52T V3 of SEQ ID NO: 633 VEGF V4 KHSAKAEBBIt-A13I-S255-M27A-L29P- BBIt-AV- +++ (SEQ ID NO: 471) S31A-F50K-V52TV4 of SEQ ID NO: 634 VEGF V5 KPSSASS BBIt-A13I-S25R-M27A-L29P- BBIt-AV-++ (SEQ ID NO: 472) S31A-I40A-F50K-V52T V5 of SEQ ID NO: 635 VEGF V6PVTKRVH BBIt-A13I-S25R-M27A-L29P- BBIt-AV- +++ (SEQ ID NO: 473)S31A-F50K-V52T V6 of SEQ ID NO: 636 VEGF Wild-type ALSYPAQ BBI sBBI −negative (SEQ ID NO: 675) SEQ ID NO: 13 control for VEGF binding FGF-5MM007 RTQPYPL BBIt-A13I-L29P-F50T-V52A MM007-Q- ++ (SEQ ID NO: 670)BBIt-FGF- 5 of SEQ ID NO: 432 FGF-5 FGFps2 TWIDSTPBBIt-A13I-L29P-F50T-V52A FGFps2- ++ (SEQ ID NO: 671) Q-BBIt- FGF-5 ofSEQ ID NO: 434 FGF-5 Wild-type ALSYPAQ BBI sBBI − negative (SEQ ID NO:675) SEQ ID NO: 13 control for FGF-5 binding TGFβ1 PEN3 PENINVLPBBIt-A40L-F50N PEN3-Q- ++ (SEQ ID NO: 672) BBIt-TGF- A40L- F50N (SEQ IDNO: 677) TGFβ1 PEN3 PENINVLP BBIt-A13I-L29P-F50T-V52A PEN3-Q- ++ (SEQ IDNO: 672) BBIt-TGF (SEQ ID NO: 443) TGFβ1 MM021W KHNVDWLBBIt-A13I-L29P-F50T-V52A MM0212- ++ (SEQ ID NO: 673) Q-BBIt- TGF (SEQ IDNO: 445) TGFβ1 WTQ WTQHIHN BBIt-A13I-L29P-F50T-V52A WTQ-Q- ++ (SEQ IDNO: 674) BBIt-TGF (SEQ ID NO: 447) TGFβ1 FGFps2 TWIDSTPBBIt-A13I-L29P-F50T-V52A FGFps2- − Negative (SEQ ID NO: 671) Q-BBIt-control for FGF-5 of TGFβ SEQ ID NO: 434 binding TNFα T1 RYWQDIPBBIt-A13I-S25R-M27A-L29P- BBIt-TNF- ++ (SEQ ID NO: 474) S31A-F50K-V52TT1 SEQ ID NO: 637 TNFα T2 APEPILA BBIt-A13I-S25R-M27A-L29P- BBIt-TNF- +(SEQ ID NO: 475) S31A-F50K-V52T T2 SEQ ID NO: 638 TNFα T3 DMINVSIBBIt-A13I-S25R-M27A-L29P- BBIt-TNF- + (SEQ ID NO: 476) S31A-F50K-V52T T3SEQ ID NO: 639 TNFα V1 SKHSQIT BBIt-A13I-S25R-M27A-L29P- BBIt-AV of −Negative (SEQ ID NO: 468) S31A-F50K-V52T SEQ ID NO: 491 control for TNFαbinding ^(•)Binding peptide used to replace the chymotrypsin loop ofBBIt scaffold. ^(▴)Amino acid sequence of the binding peptide thatreplaces amino acids 42-48 of the BBIt of SEQ ID NO: 187. *(+++)indicates an IC50 value of about 2-10 μM; (++) indicates an IC50 valueof about 10-50 μM; (+) indicates an IC50 value of about 50-250 μM; and(−) indicates an IC50 value greater than about 250 μM

These data show that modified variant BBPIs in which the chymotrypsinloop is replaced by different VEGF binding peptides, and which furthercomprise at least one amino acid substitution in the backbone of thescaffold, specifically bind their target protein i.e. VEGF. Similarly,the modified variant BBPIs in which the chymotrypsin loop is replaced byother variant peptides e.g. FGF-5, TGFβ and TNFα, also specifically bindtheir corresponding target proteins i.e. FGF, TGF and TNF. In addition,other BBPI scaffolds i.e. BBtc, BBsb3 and BBdb in which the chymotrypsinloop has been replaced by a variant peptide e.g. VEGF variant peptide,are also capable of specifically binding to the VEGF target protein.

In conclusion, these data show that modified variant BBPI scaffoldscomprising at least one amino acid substitution and carrying differentvariant peptides in place of the chymotrypsin loop retain the ability tobind the target protein that is bound by the free (ungrafted) bindingpeptide. Thus, the data indicate that different binding peptidesequences that were previously shown to bind their corresponding targetprotein can be used to replace the chymotrypsin inhibitory loop of amodified BBI protein and be expected to bind the cognate target protein.

Example 16 Identification of Peptide T-Cell Epitopes in wt BBI

The i-mune® assay was performed for the identification of peptide T-cellepitopes in wild type BBI using naïve human T-cells, as described inWO9953038A2 and Stickler et al, 2000 (J. Immunotherapy, 23(6), 654-660,[2000]). Peptides for use in the assay were prepared based on thewild-type BBI amino acid sequenceDDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDF CYEPCKPSEDDKEN(SEQ ID NO:13). From the full length amino acid sequence of BBI, 15merpeptides were synthetically prepared by Mimotopes US West (San Diego,Calif.). Each 15mer peptide sequence was designed to overlap with theprevious and subsequent 15mer, except for three residues. The 20peptides used to screen for T-cell epitopes, corresponding to wild typeBBI sequence, were:

P1 DDESSKPCCDQCACT (P1; SEQ ID NO: 649) P2 SSKPCCDQCACTKSN (P2; SEQ IDNO: 650) P3 PCCDQCACTKSNPPQ (P3; SEQ ID NO: 651) P4 DQCACTKSNPPQCRC (P4;SEQ ID NO: 652) P5 ACTKSNPPQCRCSDM (P5; SEQ ID NO: 653) P6KSNPPQCRCSDMRLN (P6; SEQ ID NO: 654) P7 PPQCRCSDMRLNSCH (P7; SEQ ID NO:655) P8 CRCSDMRLNSCHSAC (P8; SEQ ID NO: 656) P9 SDMRLNSCHSACKSC (P9; SEQID NO: 657) P10 RLNSCHSACKSCICA (P10; SEQ ID NO: 658) P11SCHSACKSCICALSY (P11; SEQ ID NO: 659) P12 SACKSCICALSYPAQ (P12; SEQ IDNO: 660) P13 KSCICALSYPAQCFC (P13; SEQ ID NO: 661) P14 ICALSYPAQCFCVDI(P14; SEQ ID NO: 662) P15 LSYPAQCFCVDITDF (P15; SEQ ID NO: 663) P16PAQCFCVDITDFCYE (P16; SEQ ID NO: 664) P17 CFCVDITDFCYEPCK (P17; SEQ IDNO: 665) P18 VDITDFCYEPCKPSE (P18; SEQ ID NO: 666) P19 TDFCYEPCKPSEDDK(P19; SEQ ID NO: 667) P20 FCYEPCKPSEDDKEN (P20; SEQ ID NO: 668)

Briefly, human CD4+ T cells were co-cultured with dendritic cells andpeptide for the intact peptide set, using the peptides shown in FigureY. Cytokine responses were averaged within each experiment as describedin Strickler et al, 2000 and a response to a peptide was tabulated aspositive if the stimulation index (SI) was greater than 2.95 (FIG. 18A).SI values for each donor were compiled for each peptide set and thepercent of donors responsive to each peptide is shown in FIG. 18B. Thepercent background response, 2.89%, was determined as the averagepercent responders over all the peptides in the set. The standarddeviation for this dataset was 2.21%. Major epitopes are defined ashaving a percentage response that is 3-fold or more than the background.Based on the data shown in FIGS. 18A and 18B, none of the peptideresponses were significant based on the statistical method for anunexposed donor population with a low background response rate.

Therefore, these data indicate that the wild type BBI molecule may beconsidered to have low immunogenicity potential in humans.

Example 17 Personal Care Compositions

In this Example, various personal care compositions comprising any ofthe modified variant BBPI compounds of the present invention areprovided as follows. In these formulations, the amounts are given aspercentages of the total composition, unless otherwise indicated. Also,unless otherwise indicated in the following formulations, theconcentration of BBI-AV (referred to as “Compound” below) ranges fromabout 0.01% to about 1.0%. In some formulations, the preferredconcentration is in the range of about 0.1% to about 0.2%, while inother formulations, the preferred concentration is in the range of about0.05% to about 0.1% (e.g., for some hair growth inhibition embodiments);from about 0.02 to 0.1% (e.g., for some skin lightening embodiments);from about 0.5% to about 1.0% (e.g., for some skin lighteningembodiments); or at concentrations greater than about 0.1% (e.g., forsome rosacea treating embodiments). Those of skill in the art know howto determine the suitable (i.e., optimum) concentration of BBI-AV foreach product. In some of the compositions provided below, the “Compound”amount is indicated as “SA.” This indicates that the formulator shoulduse the appropriate concentration of BBI-AV as indicated above herein,or as appropriate for the specific formulation.

MOISTURIZING BODYWASH (pH 7) RAW MATERIAL (INCI Designation) AmountDeionized Water QS Glycerin 4.0 PEG-6 Caprylic/Capric Glycerides 4.0Palm Kernel Fatty acids 3.0 Sodium Laureth-3 Sulphate 45.0 Cocamide MEA3.0 Sodium Lauroamphoacetate 25.0 Soybean Oil 10.0 Polyquaternium-100.70 Preservative, fragrance, color QS Compound 1000 ppm

BODY WASH RAW MATERIAL pH 8 pH 6.5 pH 7 (INCI Designation) Amount AmountAmount Deionized water QS QS QS Sodium Laureth Sulphate 12 15 8Cocamidopropyl Betaine 8 10 15 Decyl Glucoside 0 2 1 Polyquaternium-100.25 0 0 Polyquaternium-7 0 0 0.7 Preservative, fragrance, color QS QSQS Compound 250 ppm 500 ppm 1000 ppm

BODY LOTION RAW MATERIAL pH 7 pH 7 pH 7.5 pH 7 (INCI Designation) AmountAmount Amount Amount Deionized Water QS QS QS QS Glycerine 8 8 0 12Isohexadecane 3 3 3 6 Niacinamide 0 3 5 6 Isopropyl Isostearate 3 3 3 3Polyacrylamide (and) 3 3 3 3 Isoparaffin (and) Laureth-7 Petrolatum 4 44 2 Nylon 12 2 2 2.5 2.5 Dimethicone 2 2 2.5 2.5 Sucrose Polycottonseed1.5 1.5 1.5 1.5 Oil Stearyl Alcohol 97% 1 1 1 1 D Panthenol 1 1 1 1DL-alphaTocopherol 1 1 1 1 Acetate Cetyl Alcohol 95% 0.5 0.5 0.5 1Behenyl Alcohol 1 1 1 0.5 Cetearyl Alcohol (and) 0.4 0.4 0.5 0.5Cetearyl Glucoside Stearic Acid 0.15 0.15 0.15 0.15 PEG-100-Stearate0.15 0.15 0.15 0.15 Preservative, fragrance, QS QS QS QS color Compounds250 ppm 500 ppm 750 ppm 1000 ppm

ULTRA-HIGH MOISTURIZING EMULSION RAW MATERIAL pH 7 pH 7 (INCIDesignation) Amount Amount Deionized water QS QS Glycerin 12 5 PEG 400 010 Niacinamide 5 7 Isohexadecane 5 5 Dimethicone 3 2 Polyacrylamide(and) Isoparaffin (and) 3 3 Laureth-7 Isopropyl Isostearate 2 2Polymethylsilsesquioxane 2 2 Cetyl Alcohol 95% 1 1 Sucrosepolycottonseed oil 1 1 D-Panthenol 1 1 Tocopherol Acetate 1 1 StearylAlcohol 95% 0.5 0.5 Cetearyl Glucoside 0.5 0.5 Titanium dioxide 0.3 0.3Stearic Acid 0.15 0.15 PEG-100-Stearate 0.15 0.15 Preservative,fragrance, color QS QS Compound 250 ppm 100 ppm

MOISTURIZING CREAM RAW MATERIAL pH 7 pH 7 pH 7.5 (INCI Designation)Amount Amount Amount Deionized water QS QS QS Glycerine 3 5 10Petrolatum 3 3 0 Cetyl Alcohol 95% 1.5 1.5 1 Dimethicone Copolyol 2 2 2Isopropyl Palmitate 1 1 0.5 Carbopol 954 (Noveon) 0.7 0.7 0.7Dimethicone (350cs) 1 1 1 Stearyl Alcohol 97% 0.5 0.5 1 Stearic acid 0.10.1 0.1 Peg-100-stearate 0.1 0.1 0.1 Titanium Dioxide 0.3 0.3 0.3Preservative, color, fragrance QS QS QS Compound 50 ppm 250 ppm 1000 ppm

FACIAL CLEANSING EMULSION RAW MATERIAL (INCI Designation) Amount Water69.05 Disodium EDTA 0.1 Glyceryl polymethacrylate (and) Propylene glycol1.0 Glycerin 2.0 Xanthan gum 0.5 Hydroxyethyl cellulose 0.5 Tridecylneopentanoate 4.0 Isocetyl stearate 6.0 Octyl palmitate 8.0 Glyceryldilaurate 4.0 PEG-20 stearate 2.0 Glyceryl stearate (and) Laureth-23 2.0Lauryl pyrrolidone 0.5 Chamomile extract 0.2 Aloe vera (200x) 0.05Fragrance, preservative QS Compound SA

SURFACTANT-BASED FACIAL CLEANSER RAW MATERIAL (INCI Designation) AmountWater 62.55 Acrylates/Steareth-20 methacrylate copolymer 3.3 DisodiumEDTA 0.05 Glycerin 2.0 Glyceryl polymethacrylate (and) Propylene glycol(and) 0.5 PVM/MA copolymer Sodium laureth sulfate (30%) 17.5 Cetearylalcohol 1.0 Shea butter 1.0 Disodium oleamido PEG-2 sulfosuccinate 5.0Cocoamidopropyl Betaine 3.0 Sodium lauroyl sarcosinate 1.0 PEG-7glyceryl cocoate 1.0 Isodecyl oleate 1.5 Peppermint extract 0.25Eucalyptus extract 0.25 Fragrance, preservative, color, pH adjust QSCompound SA

FACIAL EXFOLIATING GEL RAW MATERIAL (INCI Designation) Amount Water64.39 Disodium EDTA 0.05 Aloe vera (200x) 0.01 Benzophenone-4 0.25Propylene glycol 1.0 Acrylates/C10-30 alkyl acrylate crosspolymer (2%)20.0 Glyceryl polymethacrylate (and) Propylene glycol 10.0 Glycerylpolymethacrylate (and) Propylene glycol (and) 1.0 PVM/MA copolymerHydrogenated jojoba oil 1.5 Fragrance, preservative, color, pH adjust QSCompound SA

FACIAL TONER RAW MATERIAL (INCI Designation) Amount Water 93.99 DisodiumEDTA 0.1 Butylene glycol 2.0 Aloe vera (200x) 0.1 Allantoin 0.1Benzophenone-4 0.5 Witch hazel extract 0.3 Propylene glycol (and)Euphrasia extract (and) 0.01 Golden seal root extract (and) Green teaextract PEG-40 hydrogenated castor oil 0.5 Quaternium-22 0.5 Sandlewoodoil 0.02 Fragrance, preservative, color, pH adjust QS Compound SA

EXFOLIATING CREAM RAW MATERIAL (INCI Designation) Amount Water 68.80Disodium EDTA 0.1 PVM/MA decadiene crosspolymer 1.0 Butylene glycol 3.0PEG-20 stearate 1.0 Glyceryl stearate (and) Laureth-23 2.0 Diisopropyladipate 2.0 Isodecyl oleate 2.0 Isocetyl stearoyl stearate 5.0 Myristylmyristate 1.0 Glyceryl dilaurate 2.0 Sodium hydroxide, 10% 2.6 Glycerylpolymethacrylate (and) Propylene glycol 5.0 Glyceryl polymethacrylate(and) Propylene glycol (and) 0.5 PVM/MA copolymer Hydrogenated jojobaoil 3.0 Fragrance, preservative, color, pH adjust QS Compound SA

FACIAL MASK RAW MATERIAL (INCI Designation) Amount Water 76.4 DisodiumEDTA 0.1 Bentonite 12.5 Potassium C12-13 Alkyl Phosphate 5.0 Propyleneglycol 4.0 Sodium Coco PG-Dimonium Chloride Phosphate 1.0 Fragrance,preservative, color, pH adjust QS Compound SA

AFTER-SHAVE BALM RAW MATERIAL (INCI Designation) Amount Water 82.12Disodium EDTA 0.1 Acrylate copolymer 2.0 Acrylate/Stareth-20methacrylate copolymer 1.0 Propylene glycol 3.0 Sodium hydroxide (10%)1.28 Glyceryl stearate (and) Cetyl alcohol (and) Stearyl alcohol 3.5(and) Behenyl alcohol (and) Palmitic acid (and) Stearic acid (and)Hydroxyethyl cetearamidopropyldimonium chloride Isocetyl stearate 1.0C12-15 alkyl lactate 1.5 Octyldodecyl stearate 3.0 Glycerylpolymethacrylate (and) Propylene glycol (and) 1.0 PVM/MA copolymerPolyquaternium-11 0.5 Fragrance, preservative, color, pH adjust QSCompound SA

EYE GEL RAW MATERIAL (INCI Designation) Amount Water 89.14VP/Acrylates/Lauryl methacrylate copolymer 0.5 Glycerin 5.0 Aminomethylpropanol 0.3 Aloe vera (200x) 0.05 Benzophenone-4 0.1 Glycerylpolymethacrylate (and) Propylene glycol (and) 0.2 PVM/MA copolymerButylene glycol (and) Water (and) Witch hazel extract 0.5 Butyleneglycol (and) Water (and) Cucumber extract 0.3 PEG-40 hydrogenated castoroil 0.01 Acrylates/Beheneth-25 methacrylate copolymer 2.4 Fragrance,preservative, color, pH adjust QS Compound SA

HIGH MELTING POINT LIPSTICK RAW MATERIAL (INCI Designation) AmountOzokerite wax 5.0 Candelilla wax 11.0 Octyl dodecanol 26.0 C30-45 alkylmethicone 5.0 Cyclomethicone 4.8 Petrolatum 3.0 Lanolin oil 9.0 Avocadooil 2.0 Oleyl alcohol 8.0 Pigment/cyclomethicone 25.0 Fragrance,preservative QS Compound SA

LIPSTICK RAW MATERIAL (INCI Designation) Amount Candelilla wax 9.1Isopropyl myristate 9.6 Lanolin 5.0 Beeswax 4.0 Paraffin (130/135) 2.0Ozokerite wax 2.5 Castor oil 53.7 Carnauba wax 1.5 Pigments 7.5 Mineraloil 4.0 Fragrance, preservative QS Compound SA

LIP GLOSS RAW MATERIAL (INCI Designation) Amount Bis-diglycerylpolyacyladipate-1 43.5 Bis-diglyceryl polyacyladipate-2 10 Glycerolricinoleate 10 Polyisobutene 1000 13 Lanolin wax 10 Candelilla wax 2.5Mica (and) titanium dioxide 3 d-Panthenol 5 Fragrance, preservative,color QS Compound SA

LIP GLOSS WITH SUNSCREEN RAW MATERIAL (INCI Designation) AmountTriisostearyl Citrate 58.4 Candelilla wax 8.0 Myristyl lactate 7.5Microcrystalline wax 5.0 Carnauba wax 2.0 Diisopropyl dimmer dilinoleate10.0 Mica (and) Bismuth oxychloride (and) Carmine 6.0 Zinc oxide(microfine) 2.0 Fragrance, preservative QS Compound SA

LIP BALM RAW MATERIAL (INCI Designation) Amount Petrolatum 47.3Isopropyl lanolate 6.0 Ozokerite wax 16.5 Candelilla wax 4.5 Diisopropyldilinoleate 25.0 Retinyl palmitate 0.5 Tocopherol acetate 0.2 Fragrance,preservative QS Compound SA

WATERPROOF MASCARA RAW MATERIAL (INCI Designation) Amount Water 49.45Propylene glycol 3.0 Triethanolamine (99%) 3.1 Acrylates/OctylacrylamineCopolymer 5.0 Diisostearoyl trimethylolpropane siloxy silicate 5.0Candelilla wax 4.5 Beeswax 5.5 Ozokerite wax 2.0 Carnauba wax 1.0 Cetylalcohol 3.0 Stearic acid 5.0 Iron oxides 11.0 Fragrance, preservative QSCompound SA

ANHYDROUS WATERPROOF MASCARA RAW MATERIAL (INCI Designation) AmountC9-11 Isoparaffin 30.95 Polyethylene 11.0 Candelilla wax 4.5Hydroxylated lanolin 0.25 Pentaerythrityl rosinate 2.0 Zinc stearate 1.0Silica silylate 1.0 Petroleum distillates (and) Quaternium-18 35.0hectorite (and) Propylene Carbonate Iron oxides 12.0 Fragrance,preservative QS Compound SA

WATER-BASED MASCARA RAW MATERIAL (INCI Designation) Amount Water 43.32Polyvinyl pyrrolidone (K30) 2.0 Hydroxyethyl cellulose 1.0Triethanolamine (99%) 2.0 Disodium EDTA 0.1 Iron Oxides 10.0 Stearicacid 4.5 Glyceryl monostearate 2.0 Beeswax 7.0 Carnauba wax 4.5Hydroxylated lanolin 1.0 Acrylates copolymer 20.0 Fragrance,preservative QS Compound SA

LIQUID EYELINER RAW MATERIAL (INCI Designation) Amount Water 50-70Gellant 0.5-1.5 Wetting agent(s) 1-3 Polyol 4-8 Colorants 10-20 Alcohol 5-10 Film former 3-8 Fragrance, preservative QS Compound SA

NAIL ENAMEL RAW MATERIAL (INCI Designation) Amount Solvent(s) 40-70Resin(s) 10-20 Plasticizer  3-12 Gellant 0-2 Colorants 0-3 Fragrance,preservative QS Compound SA

CUTICLE TREATMENT RAW MATERIAL (INCI Designation) Amount Petrolatum 34.8Beeswax 7.2 Ozokerite wax 4.3 Candelilla wax 4.0 Cocoa butter 1.0 Sheabutter 1.0 Glyceryl dilaurate 8.0 Ethylhexyl palmitate 20.0 C12-15 alkyllactate 6.0 PVP/Eicosene copolymer 3.5 Diisopropyl adipate 2.0Octinoxate 7.5 Retinyl palmitate 0.1 Tocopherol acetate 0.1 Fragrance,preservative, color, pH adjust QS Compound SA

PRESSED POWDER FORMULATIONS Pressed Eye Loose Powder Powder FoundationBlush Shadow Fillers 70-95 40-90 40-80 40-80 40-80  (e.g., talc, mica,seracite) Compression aids   0-2.5 3-5 2-5 2-7 2-10 (e.g., metallicsoaps, waxes) Texture enhancers 10-40  5-40 10-40 10-40 0-30 Colorants 2-10  2-10  5-20  2-10 1-40 (e.g., iron oxides, organic colors) Pearls 0-20  0-10 0-5  0-20 0-60 (e.g. titanated mica, bismuth oxychloride)Wet binder 0-3 2-5 2-5  3-10 3-15 (e.g., Octyldodecyl stearoyl stearate,di-PPG3 myristyl ether adipate, isocetyl stearate, cetyl dimethicone)Dry binder 0-2 2-5 2-5 3-8 3-8  (e.g., calcium silicate, kaolin)Fragrance, preservative QS QS QS QS QS Compound SA SA SA SA SA

WATER-IN-OIL FOUNDATION RAW MATERIAL (INCI Designation) AmountCyclomethicone 12.0 Dimethicone 5.0 Cyclomethicone (and) Dimethiconecopolyol 20.0 Laureth-7 0.5 Colorants (hydrophobically treated) 2.2Titanium dioxide (and) methicone 8.5 Talc (and) methicone 3.3 Water 37.2Sodium chloride 2.0 Propylene glycol 8.0 Fragrance, preservative QSCompound SA

ANHYDROUS MAKEUP STICK RAW MATERIAL (INCI Designation) Amount Ozokeritewax 5.6 Polyethylene 5.3 Glyceryl dilaurate 5.5 Isostearyl neopentanoate13.0 Octyldodecyl stearoyl stearate 12.0 Myristyl myristate 11.0Ethylhexyl methoxycinnamate 7.5 PVP/Eicosene copolymer 0.5 Tocopherolacetate 0.1 Dimethicone (and) Trimethylsiloxysilicate 8.0Cyclopentasiloxane 9.0 Mica 10.0 Talc 1.7 Titanium dioxide (and)Isopropyl titanium 8.86 triisostearate Iron oxides (and) Isopropyltitanium triisostearate 1.94 Fragrance, preservative QS Compound SA

WATER-IN-SILICONE FOUNDATION RAW MATERIAL (INCI Designation) AmountCetyl dimethicone copolyol 0.45 Polyglycerol-4 isostearate (and) Cetyldimethicone 1.75 copolyol (and) Hexyl laurate Polyalkylene polysiloxanecopolymer 0.9 Cetyl dimethicone 0.9 Beeswax 0.7 Castor wax (and)hydrogenated castor oil 0.35 Octyl palmitate 7.0 Cyclomethicone 7.95Phenyl trimethicone 2.2 Titanium dioxide (and) Caprylyl silane 7.5 Ironoxides (and) Caprylyl silane 1.1 Talc (and) Caprylyl silane 3.8Cyclomethicone 7.95 Dimethicone 1.3 Water 49.55 Sodium chloride 0.5Propylene glycol 5.3 Fragrance, preservative QS Compound SA

OIL-IN-WATER FOUNDATION RAW MATERIAL (INCI Designation) Amount Water59.85 Polyvinylpyrrolidone 5.0 Magnesium aluminum silicate 2.0 Xanthangum 0.4 Trisodium EDTA 0.05 Glyceryl polymethacrylate (and) Propyleneglycol 1.0 (and) PVM/MA copolymer Polysorbate 20 1.0 Kaolin 0.8 Butyleneglycol 4.0 Titanium dioxide 6.05 Iron oxides 1.15 Dimethicone 6.0Ethylhexyl palmitate 2.0 PEG/PPG-25/25 Dimethicone 1.0 Tocopherolacetate 0.1 Retinyl palmitate 0.1 Silica 3.0 Cyclopentasiloxane 5.0Fragrance, preservative QS Compound SA

SUNSCREEN FORMULAE RAW MATERIAL Amount (INCI Designation) SPF ~25 SPF~15 Water 52.65 71.10 PVM/MA decadiene crosspolymer 0.5 0.5 Butyleneglycol 3.0 3.0 Disodium EDTA 0.1 0.1 PEG-20 stearate 1.5 1.5 Glycerylstearate (and) Laureth-23 2.0 2.0 Isostearyl neopentanoate 1.0 1.0Ethylhexyl palmitate 2.0 2.0 Glyceryl dilaurate 0.5 0.5 Octinoxate 7.57.5 Oxybenzone 2.0 2.0 Ethylhexyl salicylate 3.0 3.0 Sodium hydroxide(10%) 1.3 1.3 Glyceryl polymethacrylate (and) Propylene glycol 3.0 3.0Glyceryl polymethacrylate (and) Propylene glycol 0.5 0.5 (and) PVM/MAcopolymer Styrene/Acrylates copolymer (27% solids) 18.45 — Fragrance,preservative QS QS Compound SA SA

VERY WATER-RESISTANT SUNSCREEN FORMULAE RAW MATERIAL Amount (INCIDesignation) SPF ~12 SPF ~22 Water 65.16 46.53 Acrylates copolymer 3.03.0 Disodium EDTA 0.1 0.1 Butylene glycol 2.0 2.0 Gylcerylpolymethacrylate (and) Propylene 1.0 1.0 glycol (and) PVM/MA copolymerButylated PVP 0.05 0.05 Glyceryl stearate (and) Behenyl alcohol (and)4.5 4.5 Palmitic acid (and) Stearic acid (and) Lecithin (and) Laurylalcohol Tricontanyl PVP 1.0 1.0 Octyl palmitate 2.0 2.0 Octinoxate 7.57.5 Oxybenzone 2.0 2.0 Ethylhexyl salicylate 3.0 3.0 Tridecylneopentanoate 3.0 3.0 Glyceryl dilaurate 0.5 0.5 Sodium hydroxide (10%)1.89 1.89 Cyclopentasiloxane 2.0 2.0 Butylene glycol 1.0 1.0Styrene/Acrylates copolymer (27% solids) 18.45 — Fragrance, preservativeQS QS Compound SA SA

WATER-IN-SILICONE SUNSCREEN RAW MATERIAL (INCI Designation) Amount CetylPEG/PPG-15/15 butyl ether dimethicone 2.0 Mineral oil 3.0 Ethylhexylpalmitate 1.0 Ethylhexyl salicylate 5.0 Hydrogenated castor oil 0.5Beeswax 0.5 Octinoxate 7.5 Polyethylene 1.0 PEG-30 dipolyhydroxystearate2.0 Cyclopentasiloxane 5.0 Dimethicone 5.0 Sodium chloride 0.6Acrylates/C12-22 alkylmethacrylate copolymer 0.5 Water 66.4 Fragrance,preservative QS Compound SA

LEAVE-ON HAIR CONDITIONER RAW MATERIAL (INCI Designation) AmountDeionized Water QS Isostearamidopropyl Morpholine Lactate 6.0Hydroxyethylcellulose 1.0 Preservative, fragrance, color QS Compound1000 ppm

CREAM RINSE (pH 4) RAW MATERIAL (INCI Designation) Amount DeionizedWater QS Behentrimonium Chloride 2.0 Trilaureth-4 Phosphate 1.5 Cetylalcohol 2.0 Citric acid QS Preservative, fragrance, color QS Compound1000 ppm

NOURISHING HAIR CONDITIONER/TREATMENT (pH 6) RAW MATERIAL (INCIDesignation) Amount Deionized Water QS Behentrimonium Methosulfate (and)Cetyl Alcohol 4.0 Wheat germ oil 1.0 Cetyl alcohol 0.5 Propylene glycol5.0 PEG-60 Lanolin 1.0 Panthenol 2.0 Lupin amino acids 1.0 CocodimoniumHydroxypropyl Hydrolyzed 1.0 Wheat Protein Fragrance, preservative,color QS Compound 1000 ppm

CONDITIONING SHAMPOO RAW MATERIAL (INCI Designation) Amount DeionizedWater QS Sodium Laureth Sulfate 30% 27.0 Cocamidopropyl Betaine 3.7Coco-Glucoside (and) Glyceryl Oleate 5.0 Coco-Glucoside (and) GlycolDistearate 3.0 (and) Glycerine Guar Hydroxypropyl Trimonium Chloride 0.1Laureth-2 1.55 Fragrance, preservative, color QS Compound 1000 ppm

ANTI-DANDRUFF SHAMPOO RAW MATERIAL (INCI Designation) Amount DeionizedWater QS Magnesium Aluminum Silicate 1.0 Hydroxypropyl Methylcellulose0.8 Sodium Olefin Sulfate 40% 35.0 Lauramide DEA 4.0 Soyamide DEA 1.0Quaternium-70 Hydrolyzed Collagen 2.0 Zinc Pyrithione 40% 4.0 Fragrance,preservative, color QS Compound 1000 ppm

CLEAR SHAMPOO RAW MATERIAL (INCI Formulations (Amounts) Designation) 1 23 4 5 Texapon 13.00  15.00  10.50  12.50  10.00  N 70 Dehyton 7.50 7.005.00 5.50 10.00  PK 45 Cetiol HE 2.00 2.50 3.50 5.00 2.30 Fragrance 0.100.10 0.10 0.10 0.10 Compound SA SA SA SA SA D-Panthenol 1.00 1.50 1.801.70 1.40 USP Preservative 0.10 0.10 0.10 0.10 0.10 Citric Acid 0.100.10 0.10 0.10 0.10 Luviquat 1.50 1.00 1.50 1.20 1.10 Ultra Care Sodium1.50 1.40 1.40 1.30 1.50 Chloride Water QS (100) QS (100) QS (100) QS(100) QS (100)

SHAMPOO RAW MATERIAL (INCI Formulations (Amounts) Designation) 1 2 3 4 5Texapon 35.00  40.00  30.00  45.00  27.00  NSO Plantacare 5.00 5.50 4.903.50 7.00 2000 Tego Betain 10.00  5.00 12.50  7.50 15.00  L7 Fragrance0.10 0.10 0.10 0.10 0.10 Compound SA SA SA SA SA D-Panthenol 0.50 1.000.80 1.50 0.50 USP Preservative 0.10 0.10 0.10 0.10 0.10 Citric Acid0.10 0.10 0.10 0.10 0.10 Rewopal 0.50 2.00 0.50 0.50 2.00 LA 3 Sodium1.50 1.50 1.50 1.50 1.50 Chloride Water QS (100) QS (100) QS (100) QS(100) QS (100)

CLEAR CONDITIONING SHAMPOO RAW MATERIAL Formulations (Amounts) (INCIDesignation) 1 2 3 4 5 Amphotensid GB 2009 10.00  15.00  20.00  12.00 17.00  Plantacare 2000 5.00 6.00 7.00 8.00 4.00 Tego Betain L7 15.00 12.00  10.00  18.00  20.00  Luviquat FC 550 0.30 0.20 0.20 0.20 0.30Fragrance 0.10 0.10 0.10 0.10 0.10 Compound SA SA SA SA SA Cremophor PS20 5.00 1.00 1.00 7.00 5.00 Preservative 0.10 0.10 0.10 0.10 0.10Rewopal LA 3 2.00 1.00 0.50 2.00 2.00 Citric Acid 0.20 0.20 0.20 0.200.20 Stepan PEG 600 DS 3.00 2.00 2.00 3.00 2.50 Water QS (100) QS (100)QS (100) QS (100) QS (100)

FOAM O/W-EMULSION Formulations RAW MATERIAL (Amounts) (INCI Designation)1 2 Stearic acid 5.00 1.00 Cetyl alcohol 5.50 Cetylstearyl alcohol 2.00PEG-40 Stearate 8.50 PEG-20 Stearate 1.00 Caprylsäure/Caprinsäuretriglyceride 4.00 2.00 C12-15 Alkylbenzoate 10.00 15.00 Cyclomethicone4.00 Dimethicone 0.50 Compound SA SA Octylisostearate 5.00 MyristylMyristate 2.00 Ceresin 1.50 Glycerine 3.00 Filter Hydroxypropyl distärkephosphate 1.00 3.50 BHT 0.02 Disodium EDTA 0.50 0.10 Parfüm,Konservierungsmittel QS QS Colorant QS QS Potassium hydroxide QS QSWater dem. QS (100) QS (100) pH adjusted to pH adjusted to 6.5-7.55.0-6.0 Emulsion 1 Emulsion 2 Gas (Stickstoff) Gas (Helium)

CONDITIONER SHAMPOO WITH PEARLESCENT Formulations RAW MATERIAL (Amounts)(INCI Designation) 1 2 3 Polyquarternium-10 0.50 0.50 0.40Sodiumlaurethsulfat 9.00 8.50 8.90 Cocoamidopropylbetain 2.50 2.60 3.00Benzophenon-4 1.50 0.50 1.00 Compound SA SA SA Pearlescent compound 2.002.50 Disodium EDTA 0.10 0.15 0.05 Preservative, Perfume, thickener QS QSQS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO Formulations RAW MATERIAL (Amounts) (INCIDesignation) 1 2 3 Polyquarternium-10 0.50 0.50 0.50 Sodiumlaurethsulfat9.00 8.50 9.50 Compound SA SA SA Benzophenon-3 1.00 1.50 0.50Imidosuccinicacid, Na 0.20 0.20 0.80 Preservative, Perfume, thickener QSQS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO WITH VOLUME EFFECT Formulations RAW MATERIAL(Amounts) (INCI Designation) 1 2 3 Natriumlaurethsulfat 10.00 10.5011.00 Ethylhexyl Methoxycinnamat 2.00 1.50 2.30 Compound SA SA SACocoamidopropylbetain 2.50 2.60 2.20 Disodium EDTA 0.01 0.10 0.01Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100)QS (100) pH adjusted to 6.0

CONDITIONING SHAMPOO WITH PEARLESCENT Formulations RAW MATERIAL(Amounts) (INCI Designation) 1 2 3 Polyquarternium-10 0.50 0.50 0.40Sodiumlaurethsulfat 9.00 8.50 8.90 Cocoamidopropylbetain 2.50 2.60 3.00Benzophenon-4 1.50 0.50 1.00 Compound SA SA SA Pearlescent compound 2.002.50 Disodium EDTA 0.10 0.15 0.05 Preservative, Perfume, thickener QS QSQS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO Formulations RAW MATERIAL (Amounts) (INCIDesignation) 1 2 3 Polyquarternium-10 0.50 0.50 0.50 Sodiumlaurethsulfat9.00 8.50 9.50 Compound SA SA SA Benzophenon-3 1.00 1.50 0.50Imidosuccinicacid, Na 0.20 0.20 0.80 Preservative, Perfume, thickener QSQS QS Water dem. QS (100) QS (100) QS (100) pH adjusted to 6.0

CLEAR CONDITIONING SHAMPOO WITH VOLUME EFFECT Formulations RAW MATERIAL(Amounts) (INCI Designation) 1 2 3 Natriumlaurethsulfate 10.00 10.5011.00 Ethylhexyl Methoxycinnamat 2.00 1.50 2.30 Compound SA SA SACocoamidopropylbetain 2.50 2.60 2.20 Disodium EDTA 0.01 0.10 0.01Preservative, Perfume, thickener QS QS QS Water dem. QS (100) QS (100)QS (100) pH adjusted to 6.0

GEL CREME Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4Acrylat/C10-30 Alkylacrylat Crosspolymer 0.40 0.35 0.40 0.35Polyacrylicacid 0.20 0.22 0.20 0.22 Xanthan Gummi 0.10 0.13 0.10 0.13Cetearylalkohol 3.00 2.50 3.00 2.50 C12-15 Alkylbenzoat 4.00 4.50 4.004.50 Caprylic/Capric Triglycerid 3.00 3.50 3.00 3.50 Aminobenzophenon(e.g., UVINUL A 2.00 1.50 0.75 1.00 PLUS ™) UVASorb K2A 3.00 EthylhexylMethoxycinnamat 3.00 1.00 Bis-Ethylhexyloxyphenol methoxyphenyl 1.502.00 Triazin Butyl Methoxydibenzoylmethan 2.00 Disodium PhenylDibenzimidazol 2.50 0.50 2.00 Tetrasulfonat Ethyhexyl Triazon 4.00 3.004.00 Octocrylen 4.00 Diethylhexyl Butamido Triazon 1.00 2.00Phenylbenzimidazol Sulfonsäure 0.50 3.00 Methylen Bis-Benzotriazolyl2.00 0.50 1.50 Tetramethylbutylphenol Ethylhexysalicylate 3.00Drometrizol Trisiloxan 0.50 Terephthaliden Dicamphor Sulfonsäure 1.501.00 Diethylhexyl-2,6-naphthalate 3.50 4.00 7.00 9.00 Titaniumdioxide-microfine 1.00 3.00 Zincoxide-microfine 0.25 Compound SA SA SASA Cyclisches Dimethylpolysiloxane 5.00 5.50 5.00 5.50 DimethiconPolydimethylsiloxane 1.00 0.60 1.00 0.60 Glycerine 1.00 1.20 1.00 1.20Sodium hydoxide QS. QS QS QS Preservative 0.30 0.23 0.30 0.23 Perfume0.20 0.20 Water QS QS QS QS (100) (100) (100) (100) pH adjusted to 6.0

O/W SUNSCREEN FORMULATION RAW MATERIAL Formulation (Amounts) (INCIDesignations) 1 2 3 4 5 6 7 Glycerin monostearate SE 0.50 1.00 3.00 1.50Glycerl Stearate Citrate 2.00 1.00 2.00 4.00 Stearic acid 3.00 2.00PEG-40 Stearate 0.50 2.00 Cetyl Phosphate 1.00 Cetearyl Sulfate 0.75Stearyl Alcohol 3.00 2.00 0.60 Cetyl Alcohol 2.50 1.10 1.50 0.60 2.00Compound SA SA SA SA SA SA SA Aminobenzophenon (e.g., UVINUL 2.00 1.500.75 1.00 2.10 4.50 5.00 A PLUS ™) UVASorb K2A EthylhexylMethoxycinnamate 5.00 6.00 8.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.502.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethane 2.00 2.00 1.50Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 0.30 TetrasulfonateEthyhexyl Triazone 4.00 3.00 4.00 2.00 Octocrylen 4.00 7.50 DiethylhexylButamido Triazon 1.00 2.00 1.00 1.00 Phenylbenzimidazol Sulfonsäure 0.503.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50Tetramethylbutylphenol Ethylhexysalicylat 3.00 5.00 DrometrizolTrisiloxan 0.50 1.00 Terephthaliden Dicamphor Sulfonic 1.50 1.00 1.000.50 Acid Diethylhexyl-2,6-naphthalat 3.50 7.00 6.00 9.00Titandioxid-microfine 1.00 3.00 3.50 1.50 Zinkoxid-microfine 0.25 2.00C12-15 Alkyl Benzoate 0.25 4.00 7.00 Dicapryl Ether 3.50 2.00Butylenglycol Dicaprylat/Dicaprat 5.00 6.00 Cocoglyceride 6.00 2.00Dimethicon 0.50 1.00 2.00 Cyclomethicone 2.00 0.50 0.50 Shea Butter 2.00PVP Hexadecen Copolymer 0.20 0.50 1.00 Glycerin 3.00 7.50 7.50 5.00 2.50Xanthan Gum 0.15 0.05 0.30 Sodium Carbomer 0.20 0.15 0.25 Vitamin EAcetat 0.60 0.23 0.70 1.00 Fucogel 1000 3.00 10.00  Glycin Soja 0.501.50 1.00 Ethylhexyloxyglycin 0.30 DMDM Hydantoin 0.60 0.40 0.20Glyacil-L 0.18 0.20 Methylparaben 0.15 0.25 0.50 Phenoxyethanol 1.000.40 0.40 0.50 0.40 Trinatrium EDTA 0.02 0.05 Iminosuccinicacid 0.251.00 Ethanol 2.00 1.50 3.00 1.20 5.00 Perfume 0.10 0.25 0.30 0.40 0.20Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

O/W SUNSCREEN FORMULATION RAW MATERIAL Formulation (Amounts) (INCIDesignations) 1 2 3 4 5 6 7 Glycerin monostearate SE 0.50 1.00 3.00 1.50Glycerl Stearate Citrate 2.00 1.00 2.00 4.00 Stearic acid 3.00 2.00PEG-40 Stearate 0.50 2.00 Cetyl Phosphate 1.00 Cetearyl Sulfate 0.75Stearyl Alcohol 3.00 2.00 0.60 Cetyl Alcohol 2.50 1.10 1.50 0.60 2.00Compound SA SA SA SA SA SA SA Aminobenzophenon (e.g., UVINUL 2.00 1.500.75 1.00 2.10 4.50 5.00 A PLUS ™) UVASorb K2A EthylhexylMethoxycinnamate 5.00 6.00 8.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.502.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethane 2.00 2.00 1.50Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 0.30 TetrasulfonateEthyhexyl Triazone 4.00 3.00 4.00 2.00 Octocrylen 4.00 7.50 DiethylhexylButamido Triazon 1.00 2.00 1.00 1.00 Phenylbenzimidazol Sulfonsäure 0.503.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50Tetramethylbutylphenol Ethylhexysalicylat 3.00 5.00 DrometrizolTrisiloxan 0.50 1.00 Terephthaliden Dicamphor Sulfonic 1.50 1.00 1.000.50 Acid Diethylhexyl-2,6-naphthalat 3.50 7.00 6.00 9.00Titandioxid-microfine 1.00 3.00 3.50 1.50 Zinkoxid-microfine 0.25 2.00C12-15 Alkyl Benzoate 0.25 4.00 7.00 Dicapryl Ether 3.50 2.00Butylenglycol Dicaprylat/Dicaprat 5.00 6.00 Cocoglyceride 6.00 2.00Dimethicon 0.50 1.00 2.00 Cyclomethicone 2.00 0.50 0.50 Shea Butter 2.00PVP Hexadecen Copolymer 0.20 0.50 1.00 Glycerin 3.00 7.50 7.50 5.00 2.50Xanthan Gum 0.15 0.05 0.30 Sodium Carbomer 0.20 0.15 0.25 Vitamin EAcetat 0.60 0.23 0.70 1.00 Fucogel 1000 3.00 10.00  Glycin Soja 0.501.50 1.00 Ethylhexyloxyglycin 0.30 DMDM Hydantoin 0.60 0.40 0.20Glyacil-L 0.18 0.20 Methylparaben 0.15 0.25 0.50 Phenoxyethanol 1.000.40 0.40 0.50 0.40 Trinatrium EDTA 0.02 0.05 Iminosuccinicacid 0.251.00 Ethanol 2.00 1.50 3.00 1.20 5.00 Perfume 0.10 0.25 0.30 0.40 0.20Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

HYDRODISPERSION RAW MATERIAL Formulations (Amounts) (INCI Designation) 12 3 4 5 Ceteaereth-20 1.00 0.50 Cetyl Alkohol 1.00 Sodium Carbomer 0.200.30 Acrylat/C10-30 Alkyl Acrylat 0.50 0.40 0.10 0.50 CrosspolymerXanthan Gummi 0.30 0.15 Compound SA SA SA SA SA Aminobenzophenon (e.g.,UVINUL 2.00 1.50 0.75 1.00 2.10 A PLUS ™) UVASorb K2A 3.50 EthylhexylMethoxycinnamat 5.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.50methoxyphenyl Triazin Butyl Methoxydibenzoylmethan 2.00 2.00 DinatriumPhenyl Dibenzimidazol 2.50 0.50 2.00 Tetrasulfonat Ethyhexyl Triazon4.00 3.00 4.00 Octocrylen 4.00 Diethylhexyl Butamido Triazon 1.00 2.001.00 Phenylbenzimidazol Sulfonsäure 0.50 3.00 MethylenBis-Benzotriazolyl 2.00 0.50 1.50 2.50 TetramethylbutylphenolEthylhexysalicylat 3.00 Drometrizol Trisiloxan 0.50 TerephthalidenDicamphor 1.50 1.00 1.00 Sulfonsäure Diethylhexyl-2,6-naphthalat 7.009.00 Titaniumdioxide-microfine 1.00 3.00 3.50 Zincoxide-microfine 0.25C12-15 Alkyl Benzoat 2.00 2.50 Dicapryl Ether 4.00 ButylenglycolDicaprylat/Dicaprat 4.00 2.00 6.00 Dicapryl Carbonat 2.00 6.00Dimethicon 0.50 1.00 Phenyltrimethicon 2.00 0.50 Shea Butter 2.00 5.00PVP Hexadecen Copolymer 0.50 0.50 1.00 Tricontanyl PVP 0.50 1.00Ethylhexylglycerin 1.00 0.80 Glycerin 3.00 7.50 7.50 8.50 Gylcin Soja1.50 1.00 Vitamin E Acetat 0.50 0.25 1.00 Alpha-Glucosilrutin 0.60 0.25Fucogel 1000 2.50 0.50 2.00 DMDM Hydantoin 0.60 0.45 0.25 Glyacil-S 0.20Methylparaben 0.50 0.25 0.15 Phenoxyethanol 0.50 0.40 1.00 TrinatriumEDTA 0.01 0.05 0.10 Ethanol 3.00 2.00 1.50 7.00 Perfume 0.20 0.05 0.40Water QS (100) QS (100) QS (100) QS (100) QS (100)

W/O SUNSCREEN EMULSION RAW MATERIAL Formulations (Amounts) (INCIDesignation) 1 2 3 4 5 Cetyldimethicon Copolyol 2.50 4.00Polyglyceryl-2-dipolyhydroxystearat 5.00 4.50PEG-30-dipolyhydroxystearat 5.00 Compound SA SA SA SA SAAminobenzophenon (e.g., 2.00 1.50 0.75 1.00 2.10 UVINUL A PLUS ™)UVASorb K2A 2.00 Ethylhexyl Methoxycinnamat 5.00 Bis-Ethylhexyloxyphenol1.50 2.00 2.50 methoxyphenyl Triazin Butyl Methoxydibenzoylmethan 2.002.00 Dinatrium Phenyl Dibenzimidazol 2.50 0.50 2.00 TetrasulfonatEthyhexyl Triazon 4.00 3.00 4.00 Octocrylen 4.00 Diethylhexyl ButamidoTriazon 1.00 2.00 1.00 Phenylbenzimidazol Sulfonsäure 0.50 3.00 MethylenBis-Benzotriazolyl 2.00 0.50 1.50 2.50 TetramethylbutylphenolEthylhexysalicylat 3.00 Drometrizol Trisiloxan 0.50 TerephthalidenDicamphor 1.50 1.00 1.00 Sulfonsäure Diethylhexyl-2,6-naphthalat 7.004.00 Titaniumdioxide-microfine 1.00 3.00 3.50 Zincoxide-microfine 0.25Mineraloil 12.00 10.00 8.00 C12-15 Alkyl Benzoat 9.00 Dicaprylyl Ether10.00 7.00 Butylenglycol Dicaprylat/Dicaprat 2.00 8.00 4.00 DicaprylylCarbonat 5.00 6.00 Dimethicon 4.00 1.00 5.00 Cyclomethicon 2.00 2.502.00 Shea Butter 3.00 Vaseline 4.50 PVP Hexadecen Copolymer 0.50 0.501.00 Ethylhexylglycerin 0.30 1.00 0.50 Glycerin 3.00 7.50 7.50 8.50Glycin Soja 1.00 1.50 1.00 MgSO4 1.00 0.50 0.50 MgCl2 1.00 0.70 VitaminE Acetat 0.50 0.25 1.00 Ascorbyl Palmitat 0.50 2.00 Fucogel 1000 3.501.00 DMDM Hydantoin 0.60 0.40 0.20 Methylparaben 0.50 0.25 0.15Phenoxyethanol 0.50 0.40 1.00 Trisodium EDTA 0.12 0.05 0.30 Ethanol 3.001.50 5.00 Perfume 0.20 0.40 0.35 Water QS (100) QS (100) QS (100) QS(100) QS (100)

STICKS Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4Caprylic/Capric Triglycerid 12.00  10.00  6.00 Octyldodecanol 7.0014.00  8.00 3.00 Butylene Glycol 12.00  Dicaprylat/DicapratPentaerythrityl Tetraisostearat 10.00  6.00 8.00 7.00 Polyglyceryl-3Diisostearat 2.50 Bis-Diglyceryl Polyacyladipate-2 9.00 8.00 10.00  8.00Cetearyl Alcohol 8.00 11.00  9.00 7.00 Myristyl Myristate 3.50 3.00 4.003.00 Beeswax 5.00 5.00 6.00 6.00 Cera Carnauba 1.50 2.00 2.00 1.50 CeraAlba 0.50 0.50 0.50 0.40 C16-40 Alkyl Stearat 1.50 1.50 1.50 Compound SASA SA SA Aminobenzophenon (e.g., 2.00 1.50 0.75 9.00 UVINUL A PLUS ™)UVASorb K2A 2.00 4.00 Ethylhexyl Methoxycinnamat 3.00Bis-Ethylhexyloxyphenol 1.50 2.00 methoxyphenyl Triazin ButylMethoxydibenzoylmethan 2.00 Dinatrium Phenyl Dibenzimidazol 2.50 0.502.00 Tetrasulfonat Ethyhexyl Triazon 4.00 3.00 4.00 Octocrylen 4.00Diethylhexyl Butamido Triazon 1.00 2.00 Phenylbenzimidazol Sulfonsäure0.50 3.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50Tetramethylbutylphenol Ethylhexysalicylat 3.00 Drometrizol Trisiloxan0.50 Terephthaliden Dicamphor 1.50 1.00 SulfonsäureDiethylhexyl-2,6-naphthalat 7.00 Titaniumdioxide-microfine 1.00 3.00Zincoxide-microfine 0.25 Vitamin E Acetat 0.50 1.00 Ascorbyl Palmitat0.05 0.05 Buxux Chinensis 2.00 1.00 1.00 Perfume, BHT 0.10 0.25 0,35Ricinus Communis QS QS QS QS (100) (100) (100) (100)

PIT-EMULSION RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 23 4 5 6 7 8 Glycerinmonostearat SE 0.50 2.00 3.00 5.00 0.50 4.00Glyceryl Isostearat 3.50 4.00 2.00 Isoceteth-20 0.50 2.00 Ceteareth-125.00 1.00 3.50 5.00 Ceteareth-20 5.00 1.00 3.50 PEG-100 Stearat 2.802.30 3.30 Cetyl Alkohol 5.20 1.20 1.00 1.30 0.50 0.30 Cetyl Palmitat2.50 1.20 1.50 0.50 1.50 Cetyl Dimethicon Copolyol 0.50 1.00Polyglyceryl-2 0.75 0.30 Compound SA SA SA SA SA SA SA SAAminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 4.50 5.00 2.10 APLUS ™) UVASorb K2A 4.00 1.50 Ethylhexyl Methoxycinnamat 5.00 6.00 8.005.00 Bis-Ethylhexyloxyphenol 1.50 2.00 2.50 2.50 2.50 methoxyphenylTriazin Butyl Methoxydibenzoylmethan 2.00 2.00 1.50 2.00 DinatriumPhenyl Dibenzimidazol 2.50 0.50 2.00 0.30 Tetrasulfonat EthyhexylTriazon 4.00 3.00 4.00 2.00 Octocrylen 4.00 7.50 Diethylhexyl ButamidoTriazon 1.00 2.00 1.00 1.00 1.00 Phenylbenzimidazol Sulfonsäure 0.503.00 Methylen Bis-Benzotriazolyl 2.00 0.50 1.50 2.50 2.50Tetramethylbutylphenol Ethylhexysalicylat 3.00 5.00 DrometrizolTrisiloxan 0.50 1.00 Terephthaliden Dicamphor 1.50 1.00 1.00 0.50 1.00Sulfonsäure Diethylhexyl-2,6-naphthalat 7.00 10.00  7.50 8.00Titandioxid-microfine 1.00 3.00 3.50 1.50 3.50 Zinkoxid-microfine 0.252.00 C12-15 Alkyl Benzoat 3.50 6.35 0.10 Cocoglyceride 3.00 3.00 1.00Dicapryl Ether 4.50 Dicaprylyl Carbonat 4.30 3.00 7.00 Dibutyl Adipate0.50 0.30 Phenyltrimethicone 2.00 3.50 2.00 Cyclomethicon 3.00 EthylGalaktomannan 0.50 2.00 Hydrierte Coco-Glyceride 3.00 4.00 Abil Wax 24401.50 2.00 PVP Hexadecen Copolymer 1.00 1.20 Glycerin 4.00 6.00 5.00 8.0010.00  Vitamin E Acetat 0.20 0.30 0.40 0.30 Shea Butter 2.00 3.60 2.00Iodopropyl Butylcarbamat 0.12 0.20 Fucogel 1000 0.10 DMDM Hydantoin 0.100.12 0.13 Methylparaben 0.50 0.30 0.35 Phenoxyethanol 0.50 0.40 1.00Octoxyglycerin 0.30 1.00 0.35 Ethanol 2.00 2.00 5.00 Trinatrium EDTA0.40 0.15 0.20 Perfume 0.20 0.20 0.24 0.16 0.10 0.10 Water QS (100) QS(100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

GEL CREME Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4Acrylat/C10-30 0.40 0.35 0.40 0.35 alkylacrylat crosspolymer Polyacrylicacid 0.20 0.22 0.20 0.22 Luvigel EM 1.50 2.50 2.80 3.50 Xanthan gum 0.100.13 0.10 0.13 Cetearylalkohol 3.00 2.50 3.00 2.50 C12-15 Alkylbenzoate4.00 4.50 4.00 4.50 Caprylic/Capric 3.00 3.50 3.00 3.50 TriglycerideTitan dioxide-microfine 1.00 1.50 Zinc oxide-microfine 2.00 0.25Compound SA SA SA SA Dihydroxyacetone 3.00 5.00 Cyclisches 5.00 5.505.00 5.50 Dimethylpolysiloxan Dimethicon 1.00 0.60 1.00 0.60Polydimethylsiloxan Glycerine 1.00 1.20 1.00 1.20 Natrium hydroxide QSQS QS QS Preservatives 0.30 0.23 0.30 0.23 Perfume 0.20 0.20 Water QS(100) QS (100) QS (100) QS (100) pH adjusted to 6.0

O/W SELF TANNER FORMULATIONS RAW MATERIAL Formulations (Amounts) (INCIDesignation) 1 2 3 4 5 6 7 Glycerin monostearate 0.50 1.00 3.00 1.50 SEGlycerlstearate citrate 2.00 1.00 2.00 4.00 Stearic acid 3.00 2.00PEG-40 stearate 0.50 2.00 Cetyl phosphate 1.00 Cetearyl sulfate 0.75Stearyl alcohol 3.00 2.00 0.60 Cetyl alcohol 2.50 1.10 1.50 0.60 2.00Compound SA SA SA SA SA SA SA Dihydroxy acetone 3.00 5.00 4   Titaniumdioxide- 1.00 1.50 1.50 microfine Zinc oxide-microfine 0.25 2.00 C12-15Alkyl benzoate 0.25 4.00 7.00 Dicapryl ether 3.50 2.00 Butylenglycol5.00 6.00 Dicaprylaet/Dicaprat Cocoglyceride 6.00 2.00 Dimethicon 0.501.00 2.00 Cyclomethicon 2.00 0.50 0.50 Shea butter 2.00 PVP hexadecen0.20 0.50 1.00 copolymer Glycerin 3.00 7.50 7.50 5.00 2.50 Xanthan gum0.15 0.05 0.30 Sodium carbomer 0.20 0.15 0.25 Vitamin E acetate 0.600.23 0.70 1.00 Fucogel 1000 3.00 10.00 Glycin Soja 0.50 1.50 1.00Ethylhexyloxy glycin 0.30 DMDM hydantoin 0.60 0.40 0.20 Glyacil-L 0.180.20 Methylparaben 0.15 0.25 0.50 Phenoxyethanol 1.00 0.40 0.40 0.500.40 Trinatrium EDTA 0.02 0.05 Iminobernsteinsäure 0.25 1.00 Ethanol2.00 1.50 3.00 1.20 5.00 Perfume 0.10 0.25 0.30 0.40 0.20 Water QS (100)QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

O/W MAKE UP RAW MATERIAL Formulations (Amounts) (INCI Desigation) 1 2 34 5 6 7 Glycerinmonostearat 0.50 1.00 3.00 1.50 SE Glycerl StearatCitrat 2.00 1.00 2.00 4.00 Stearicacid 3.00 2.00 PEG-40 Stearat 0.502.00 Cetyl Phosphat 1.00 Cetearyl Sulfat 0.75 Stearyl Alkohol 3.00 2.000.60 Cetyl Alkohol 2.50 1.10 1.50 0.60 2.00 Compound SA SA SA SA SA SASA Titaniumoxide 10.00 12.00 9.00 8.50 11.00 9.50 10.00 Ironoxide 2.004.00 3.00 5.00 3.40 6.00 4.40 Zincoxide 4.00 2.00 3.00 C12-15 AlkylBenzoat 0.25 4.00 7.00 Dicapryl Ether 3.50 2.00 Butylenglycol 5.00 6.00Dicaprylat/Dicaprat Cocoglyceride 6.00 2.00 Dimethicon 0.50 1.00 2.00Cyclomethicon 2.00 0.50 0.50 Shea Butter 2.00 PVP Hexadecen 0.20 0.501.00 Copolymer Glycerin 3.00 7.50 7.50 5.00 2.50 Xanthan Gummi 0.15 0.050.30 Sodium Carbomer 0.20 0.15 0.25 Vitamin E Acetat 0.60 0.23 0.70 1.00Glycin Soja 0.50 1.50 1.00 Ethylhexyloxyglycin 0.30 DMDM Hydantoin 0.600.40 0.20 Glyacil-L 0.18 0.20 Methylparaben 0.15 0.25 0.50Phenoxyethanol 1.00 0.40 0.40 0.50 0.40 Trinatrium EDTA 0.02 0.05Iminosuccinicacid 0.25 1.00 Ethanol 2.00 1.50 3.00 1.20 5.00 Perfume0.10 0.25 0.30 0.40 0.20 Water QS (100) QS (100) QS (100) QS (100) QS(100) QS (100) QS (100)

SELF TANNER HYDRODISPERSION RAW MATERIAL Formulations (Amounts) (INCIDesignation) 1 2 3 4 5 Ceteaereth-20 1.00 0.50 Cetyl Alkohol 1.00Luvigel EM 2.00 2.50 2.00 Acrylat/C10-30 Alkyl Acrylat 0.50 0.40 0.100.50 Crosspolymer Xanthan Gummi 0.30 0.15 Compound SA SA SA SA SADihydroxyaceton 3.00 5.00 Aminobenzophenon (e.g., UVINUL 2.00 1.50 0.751.00 2.10 A PLUS ™) Titandioxid-microfine 1.00 1.00 1.00Zinkoxid-microfine 1.90 0.25 C12-15 Alkyl Benzoat 2.00 2.50 DicaprylEther 4.00 Butylenglycol Dicaprylat/Dicaprat 4.00 2.00 6.00 DicaprylCarbonat 2.00 6.00 Dimethicon 0.50 1.00 Phenyltrimethicon 2.00 0.50 SheaButter 2.00 5.00 PVP Hexadecen Copolymer 0.50 0.50 1.00 Tricontanyl PVP0.50 1.00 Ethylhexylglycerin 1.00 0.80 Glycerin 3.00 7.50 7.50 8.50Gylcin Soja 1.50 1.00 Vitamin E Acetat 0.50 0.25 1.00Alpha-Glucosilrutin 0.60 0.25 DMDM Hydantoin 0.60 0.45 0.25 Glyacil-S0.20 Methylparaben 0.50 0.25 0.15 Phenoxyethanol 0.50 0.40 1.00Trinatrium EDTA 0.01 0.05 0.10 Ethanol 3.00 2.00 1.50 7.00 Parfüm 0.200.05 0.40 Water QS (100) QS (100) QS (100) QS (100) QS (100)

AFTER SUN HYDRODISPERSION Formulations (Amounts) 1 2 3 4 5 Ceteaereth-201.00 0.50 Cetyl Alkohol 1.00 Luvigel EM 2.00 2.50 2.00 Acrylat/C10-30Alkyl Acrylat 0.50 0.30 0.40 0.10 0.50 Crosspolymer Xanthan Gummi 0.300.15 Compound SA SA SA SA SA C12-15 Alkyl Benzoat 2.00 2.50 DicaprylEther 4.00 Butylenglycol 4.00 2.00 6.00 Dicaprylat/Dicaprat DicaprylCarbonat 2.00 6.00 Dimethicon 0.50 1.00 Phenyltrimethicon 2.00 0.50Tricontanyl PVP 0.50 1.00 Ethylhexylglycerin 1.00 0.80 Glycerin 3.007.50 7.50 8.50 Gylcin Soja 1.50 1.00 Vitamin E Acetat 0.50 0.25 1.00Alpha-Glucosilrutin 0.60 0.25 Trinatrium EDTA 0.01 0.05 0.10 Ethanol1.00 10.00  8.00 12.00  9.00 Perfume 0.20 0.05 0.40 Water QS (100) QS(100) QS (100) QS (100) QS (100)

WO-EMULSIONS RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 23 4 5 Cetyldimethicon Copolyol 2.50 4.00Polyglyceryl-2-dipolyhydroxystearat 5.00 4.50PEG-30-dipolyhydroxystearat 5.00 Compound SA SA SA SA SAAminobenzophenon (e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 A PLUS ™)Titaniumdioxide-microfine 1.00 3.00 3.50 Zincoxide-microfine 0.90 0.25Mineralöl 12.00 10.00 8.00 C12-15 Alkyl Benzoat 9.00 Dicaprylyl Ether10.00 7.00 Butylenglycol Dicaprylat/Dicaprat 2.00 8.00 4.00 DicaprylylCarbonat 5.00 6.00 Dimethicon 4.00 1.00 5.00 Cyclomethicon 2.00 25.002.00 Shea Butter 3.00 Vaseline 4.50 PVP Hexadecen Copolymer 0.50 0.501.00 Ethylhexylglycerin 0.30 1.00 0.50 Glycerin 3.00 7.50 7.50 8.50Glycin Soja 1.00 1.50 1.00 MgSO4 1.00 0.50 0.50 MgCl2 1.00 0.70 VitaminE Acetat 0.50 0.25 1.00 Ascorbyl Palmitat 0.50 2.00 Fucogel 1000 3.507.00 DMDM Hydantoin 0.60 0.40 0.20 Methylparaben 0.50 0.25 0.15Phenoxyethanol 0.50 0.40 1.00 Trinatrium EDTA 0.12 0.05 0.30 Ethanol3.00 1.50 5.00 Perfume 0.20 0.40 0.35 Water QS (100) QS (100) QS (100)QS (100) QS (100)

SOLID STABILIZED EMULSIONS (PICKERING EMULSIONS) RAW MATERIALFormulations (Amounts) (INCI Designation) 1 2 3 4 5 Mineral oil 16.0016.00 Octyldodecanol 9.00 9.00 5.00 Caprylic/Capric Triglycerid 9.009.00 6.00 C12-15 Alkyl Benzoat 5.00 8.00 Butylen GlycolDicaprylat/Dicaprat 8.00 Dicaprylyl Ether 9.00 4.00 Dicaprylyl Carbonat9.00 Hydroxyoctacosanyl Hydroxystearat 2.00 2.00 2.20 2.50 1.50Disteardimonium Hectorit 1.00 0.75 0.50 0.25 Cera Microcristallina +Paraffinum 0.35 5.00 Liquidum Hydroxypropyl Methylcellulose 0.10 0.05Dimethicon 3.00 Compound SA SA SA SA SA Titaniumdioxide + Alumina + 3.00Simethicon + Aqua Titaniumdioxide + 2.00 4.00 2.00 4.00Trimethoxycaprylylsilan Silica Dimethyl Silylat 2.50 6.00 2.50 Bornitrid1.00 Stärke/-Natriummetaphosphat- 2.00 Polymer Tapioca Stärke 0.50Sodium Chlorid 5.00 7.00 8.50 3.00 4.50 Glycerin 1.00 Trinatrium EDTA1.00 1.00 1.00 1.00 1.00 Vitamin E Acetat 5.00 10.00 3.00 6.00 10.00Ascorbyl Palmitat 1.00 1.00 1.00 Methylparaben 0.60 0.20 Propylparaben0.20 Phenoxyethanol 0.20 Hexamidin Diisethionat 0.40 0.50 0.40Diazolidinyl Harnstoff 0.08 Ethanol 0.23 0.20 Perfume 5.00 3.00 4.00Water 0.20 0.30 0.10 QS (100) QS (100) QS (100) QS (100) QS (100)

STICKS Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4Caprylic/Capric Triglycerid 12.00  10.00  6.00 Octyldodecanol 7.0014.00  8.00 3.00 Butylene Glycol 12.00  Dicaprylat/DicapratPentaerythrityl Tetraisostearat 10.00  6.00 8.00 7.00 Polyglyceryl-3Diisostearat 2.50 Bis-Diglyceryl Polyacyladipate-2 9.00 8.00 10.00  8.00Cetearyl Alcohol 8.00 11.00  9.00 7.00 Myristyl Myristate 3.50 3.00 4.003.00 Beeswax 5.00 5.00 6.00 6.00 Cera Carnauba 1.50 2.00 2.00 1.50 CeraAlba 0.50 0.50 0.50 0.40 C16-40 Alkyl Stearat 1.50 1.50 1.50 Compound SASA SA SA Aminobenzophenon (e.g., 2.00 1.50 0.75 9.00 UVINUL A PLUS ™)Titaniumdioxide-microfine 1.00 3.00 Zincoxide-microfine 1.00 0.25Vitamin E Acetat 0.50 1.00 Ascorbyl Palmitat 0.05 0.05 Buxux Chinensis2.00 1.00 1.00 Perfume, BHT 0.10 0.25 0.35 Ricinus Communis QS QS QS QS(100) (100) (100) (100)

SELF TANNER PIT-EMULSION Formulations (Amounts) 1 2 3 4 5 6 7 8Glycerinmonostearat SE 0.50 2.00 3.00 5.00 0.50 4.00 Glyceryl Isostearat3.50 4.00 2.00 Isoceteth-20 0.50 2.00 Ceteareth-12 5.00 1.00 3.50 5.00Ceteareth-20 5.00 1.00 3.50 PEG-100 Stearat 2.80 2.30 3.30 Cetyl Alkohol5.20 1.20 1.00 1.30 0.50 0.30 Cetyl Palmitat 2.50 1.20 1.50 0.50 1.50Cetyl Dimethicon Copolyol 0.50 1.00 Polyglyceryl-2 0.75 0.30 Compound SASA SA SA SA SA SA SA Dihydroxyaceton 3.00 5.00 4.00 Aminobenzophenon(e.g., UVINUL 2.00 1.50 0.75 1.00 2.10 4.50 5.00 2.10 A PLUS ™)Titandioxide-microfine 1.00 1.50 3.50 1.50 1.00 Zinkoxide-microfine 1.000.25 2.00 1.50 C12-15 Alkyl Benzoat 3.50 6.35 0.10 Cocoglyceride 3.003.00 1.00 Dicapryl Ether 4.50 Dicaprylyl Carbonat 4.30 3.00 7.00 DibutylAdipate 0.50 0.30 Phenyltrimethicone 2.00 3.50 2.00 Cyclomethicon 3.00Ethyl Galaktomannan 0.50 2.00 Hydrogenated CoCo-Glyceride 3.00 4.00 AbilWax 2440 1.50 2.00 PVP Hexadecen Copolymer 1.00 1.20 Glycerin 4.00 6.005.00 8.00 10.00  Vitamin E Acetat 0.20 0.30 0.40 0.30 Shea Butter 2.003.60 2.00 Iodopropyl Butylcarbamat 0.12 0.20 DMDM Hydantoin 0.10 0.120.13 Methylparaben 0.50 0.30 0.35 Phenoxyethanol 0.50 0.40 1.00Octoxyglycerin 0.30 1.00 0.35 Ethanol 2.00 2.00 5.00 Trinatrium EDTA0.40 0.15 0.20 Perfume 0.20 0.20 0.24 0.16 0.10 0.10 Water QS (100) QS(100) QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)

OILGELS Formulations RAW MATERIAL (Amounts) (INCI Designation) 1 2 3 4Caprylic/Capric 12.00 10.00 6.00 Triglycerid Octyldodecanol 7.00 14.008.00 3.00 Butylene Glycol 12.00 Dicaprylat/Dicaprat Pentaerythrityl10.00 6.00 8.00 7.00 Tetraisostearat Polyglyceryl-3 2.50 DiisostearatBis-Diglyceryl 9.00 8.00 10.00 8.00 Polyacyladipate-2 Myristyl Myristate3.50 3.00 4.00 3.00 Bentone-34 5.00 5.00 6.00 6.00 Propylencarbonat15.00 20.00 18.00 19.50 Compound SA SA SA SA Vitamin E Acetat 0.50 1.00Ascorbyl Palmitat 0.05 0.05 Buxux Chinensis 2.00 1.00 1.00 Perfume, BHT0.10 0.25 0.35 Ricinus Communis QS (100) QS (100) QS (100) QS (100)

In still further embodiments, the present invention comprises at leastone inorganic pigment. In some preferred embodiments, these inorganicpigments are based on metaloxides and/or other water slightly soluble orinsoluble metal compounds, including but not limited to compounds suchas zinc oxides (ZnO), titanium (TiO₂), iron (e.g., Fe₂O₃), zirconium(ZrO₂), silica (SiO₂), manganese (e.g., MnO), aluminium (Al₂O₃), cer(e.g., Ce₂O₃), and mixed oxides of these oxides, as well as blendsthereof. In some embodiments, the metaloxides are microfine grade, whilein other embodiments, the metaloxides are pigment grade. In furtherembodiments, the metaloxides are a mixture of microfine and pigmentgrades.

In additional embodiments, the inorganic pigments are coated (i.e., theyare treated on the surface). In some particularly preferred embodiments,the surface is coated with a thin, hydrophobic film. In some otherparticularly preferred embodiments, the surface is coated with a thin,hydrophilic film. In yet additional embodiments, the present inventionprovides compositions comprising various make ups and make upconstituents. For example, in some embodiments, the present inventionprovides various dyes and/or pigments. In some embodiments, usefulpigments include, but are not limited to titanium dioxide, mica, ironoxides (e.g. Fe₂O₃, Fe₃O₄, FeO(OH), etc.) and/or stannous oxide. Thepresent invention further provides colorants, including but not limitedto carmine, blue, chromooxide, ultramarine and/or purple manganese. Thecolorants and pigments of some most preferred embodiments are known tothose in the art and provided previously (See e.g., Colour Index Nummern(CIN), Rowe Colour Index, 3^(rd) ed., Society of Dyers and Colourists,Bradford, England [1971]).

In additional embodiments, pearlescent pigments based on mica/metaloxidefind use, as described above. However, it is not intended that thepresent invention be limited to these particular pigments, as additionalpearlescent pigments find use in various embodiments of the presentinvention.

The following formulations provide additional examples of the use of thepresent invention.

RAW MATERIAL Formulation (Amounts) (INCI Designation 1 2 3 4 5 SodiumCarbomer 0.2 Acrylates/C₁₀-C₃₀ Alkyl Acrylate 0.3 0.2 0.6 CrosspolymerHydroxypropyl Cellulose 1.0 1.50 Xanthan Gummi 0.6 0.2 1.0 1.0 Compound0.5 0.1 0.01 0.01 1.0 Dioctyl Butamidotriazon 2.0 2.0 1.0 EthylhexylTriazon 4.0 4.0 5.0 Aniso Triazin 1.0 0.5 2.0 2.5 Bisoctyltriazol 6.0Drometrizole Trisiloxane PhenylbenzmidazSulfonicacid 2.0 1.0Bisimidazylate 1.0 Terephthalylidene Dicamphor 0.2 Sulfonic AcidEthylhexyl Methoxycinnamat 7.5 10.0 5.0 Octocrylen 5.0 Dimethicone- 4.0diethylbenzalmalonate Ethylhexyl Salicylate Homosalate ButylMethoxydibenzoylmethan 1.0 1.0 4.0 Titan dioxide 1.0 4.0 Zinc oxide 4.0Caprylic/Capric Triglycerid 2.0 Hydrogenated Coco- 3.0 Glyceride C12-15Alkyl Benzoat 2.0 2.5 3.0 Dicaprylyl Ether 4.0 Butylenglycol Dicaprylat/4.0 2.0 6.0 Dicaprat Dicaprylyl Carbonat 2.0 Cetyl Dimethicon 2.0 0.51.0 Shea Butter 2.0 PVP Hexadecen Copolymer 0.5 0.05 0.5 Glycerin 3.07.5 7.5 2.5 Tocopherol 0.5 0.75 0.2 Trisodium EDTA 1.0 0.5 0.5 1.0 1.5Natriumcitrat 0.2 Zitronensäure 0.1 0.1 0.1 DMDM Hydantoin 0.6 0.2Methylparaben 0.5 0.3 0.15 Phenoxyethanol 0.5 0.4 0.4 1.0 0.60 Ethanol3.0 2.0 3.0 1.0 Perfume 0.2 0.2 0.2 Water QS (100) QS (100) QS (100) QS(100) QS (100)

RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 SodiumCarbomer 0.5 1.5 Acrylates/C₁₀-C₃₀ Alkyl 0.4 0.1 0.75 AcrylateCrosspolymer Hydroxypropyl Cellulose 0.5 0.25 Xanthan Gummi 0.2 0.4Compound 0.5 0.1 0.01 0.01 1.0 Dioctyl Butamidotriazon 1.0 2.0Ethylhexyl Triazon 2.0 2.0 Aniso Triazin 1.0 0.2 3.0 1.0 Bisoctyltriazol8.0 Drometrizole Trisiloxane 4.0 Phenylbenzmidazole 1.5 SulfonicacidBisimidazylate 1.5 Terephthalylidene Dicamphor 0.5 Sulfonic AcidEthylhexyl Methoxycinnamat 7.5 5.0 10.0 Octocrylen 10.0 5.0 5.0Dimethicone- 2.5 diethylbenzalmalonate Ethylhexyl Salicylate 3.5 5.0Homosalate 4.0 Butyl 0.5 Methoxydibenzoylmethan Titandioxide 1.5 2.0 1.02.5 Zincoxide 1.0 0.5 Caprylic/Capric Triglycerid HydrogenierteCoco-Glyceride C12-15 Alkyl Benzoat 5.0 Dicaprylyl Ether 7.5Butylenglycol Dicaprylat/Dicaprat Dicaprylyl Carbonat 7.5 CetylDimethicon Shea Butter 3.0 PVP Hexadecen Copolymer 0.5 0.75 1.0 Glycerin5.0 10.0 Tocopherol 0.3 1.5 1.0 Trisodium EDTA 0.5 0.1 0.5 Natriumcitrat0.3 Zitronensäure 0.15 DMDM Hydantoin 0.3 0.15 Methylparaben 0.4Phenoxyethanol 1.0 Ethanol 7.5 5.0 7.0 Perfume 0.25 0.2 Water QS (100)QS (100) QS (100) QS (100) QS (100)

RAW MATERIAL Formulations (Amounts) (INCI Designation) 1 2 3 4 5 6Sodium Carbomer 0.5 1.5 1.0 0.5 Acrylates/C₁₀-C₃₀ Alkyl 1.0 0.75 1.0Acrylate Crosspolymer Hydroxypropyl Cellulose 0.4 1.0 1.0 Xanthan Gummi0.6 0.2 1.0 1.0 BBI 4.0 0.5 3.0 2.0 4.0 1.5 Dioctyl Butamidotriazon 2.02.0 2.0 1.0 Ethylhexyl Triazon 4.0 5.0 4.0 Aniso Triazin 1.0 1.0 2.5 1.0Bisoctyltriazol 4.0 Drometrizole Trisiloxane 3.0 Phenylbenzmidazole 2.01.0 Sulfonicacid Bisimidazylate 1.5 3.5 Terephthalylidene 0.2 1.0Dicamphor Sulfonic Acid Ethylhexyl 10.0 5.0 Methoxycinnamat Octocrylen10.0 5.0 Dimethicone- 4.0 diethylbenzalmalonate Ethylhexyl 5.0Salicylate Homosalate 5.0 Butyl Methoxy- 1.0 1.0 4.0 0.5 dibenzoylmethanTitandioxide 1.0 4.0 1.5 Zincoxide 4.0 Caprylic/Capric 2.0 TriglyceridParaffinöl 1.0 C₁₂-C₁₅ Alkyl 2.0 2.5 3.0 Benzoat Dicaprylyl Ether 4.0Isohexadecen 4.0 2.0 6.0 Dicaprylyl 2.0 Carbonat Dibutyl Adipat 2.0 0.51.0 Cylomethicon 3.0 Jojobaöl 2.0 PVP Hexadecen 0.5 0.05 0.5 0.5Copolymer Butylen Glycol 3.0 7.5 7.5 2.5 5.0 Ascorbyl-Palmitate 0.5 0.750.2 0.3 Octoxyglycerin 1.0 0.5 1.0 Glycin Soja 2.0 1.5 Trisodium EDTA1.0 0.5 0.5 1.5 0.5 Caustic acid 1.0 0.2 0.25 Iodopropyl 0.6 0.2Butylcarbamat Phenoxyethanol 0.4 1.0 Ethanol 5.0 2.0 7.0 Perfume 0.2 0.20.2 Water QS (100) QS (100) QS (100) QS (100) QS (100) QS (100)The following formula provides an example of an after-shave productcomprising the BBI-AV of the present invention.

AFTER SHAVE LOTION % Ingredient INCI A 10.0 Luvitol EHO CetearylEthylhexanoate 5.0 Vitamin E Acetate Tocopheryl Acetate 1.0 Bisabololrac. Bisabolol 0.1 Perfume 0.3 Carbopol Ultrez 21 Acrylates/C10-30 AlkylAcrylate Crosspolymer B 15.0 Ethanol Alcohol 1.0 D-Panthenol USPPanthenol 3.0 Glyerin 87% Glycerin 0.1 Triethanolamine CareTriethanolamine SA Compound QS Water dem. Aqua dem.Production: Weigh out the components of Phase A and mix them. DissolvePhase B, stir it into Phase A and homogenize well.Measure Values:

Viscosity: 18 500 mPa s Brookfield RVD II+

pH value: 5.8

The following formula provides an example of an after-shave productcomprising the BBI-AV of the present invention.

PRE SHAVE % Ingredient INCI A 80 Ethanol Alcohol 3.0 Vitamin E AcetateTocopheryl Acetate 1.0 Bisabolol rac. Bisabolol 0.2 Perfume 0.1 MentholMenthol 4.0 Luvitol EHO Cetearyl Ethylhexanoate 2.0 Eutanol GOctyldodecanol 2.0 Miglyol 812 Caprylic/Capric Triglyceride 2.0D-Panthenol USP Panthenol 2.0 Whitch Hazel Distillate HamamelisVirginiana (Whitch Hazel) Distillate 2.0 Jojoba Oil Simmondsia Chinensis(Jojoba) Seed Oil SA CompoundProduction: Weigh out the components of Phase A and dissolve themclearly.

The after-shave and pre-shave formula provided above contain sufficientBBI-AV (Compound) to provide the desired effect(s). In some embodiments,the concentration of BBI-AV is in the range of about 1,000 ppm to about10,000 ppm. In the following formulations, typical concentrations ofBBI-AV used range from about 100 ppm to about 1,000 ppm or from about1,000 ppm to about 10,000 ppm. However, it is not intended that thepresent invention be limited to this specific concentration range, asother concentrations find use in other embodiments of the presentinvention.

The following formula provides an example of an after-sun productcomprising the BBI-AV of the present invention.

AFTER SUN LOTION % Ingredient INCI A 0.4 Carbopol 1342 Acrylates/C10-30Alkyl Acrylate Crosspolymer 15.0 Luvitol EHO Cetearyl Ethylhexanoate 0.2Bisabolol rac. Bisabolol 1.0 Vitamin E Acetate Tocopheryl Acetate q.s.Perfume B 1.0 D-Panthenol USP Panthenol 15.0 Ethanol 96% Alcohol 3.0Glycerin 87% Glycerin SA Compound 64.2 Water dem. Aqua dem. C 0.2Triethanolamine Care TriethanolamineProduction: Mix the components of Phase A. Dissolve Phase B and stir itinto Phase A whilst homogenizing. Neutralize with Phase C and homogenizeagain.Measure Values:

Viscosity: 7 500 mPa s Haake Viscotester VT-02

pH value: 6.0

The following formula provides an example of a facial cleanser productcomprising the BBI-AV of the present invention.

FACIAL CLEANSER % Ingredient INCI A 10.0  Luvitol EHO CetearylEthylhexanoate 10.0  Miglyol 812 Caprylic/Capric Triglyceride 1.5 DowCorning 345 Fluid Cyclopentasiloxane, Cyclohexasilosane 2.0 Cremophor CO40 PEG-40 Hydrogenated Castor Oil B 3.5 Luvigel EM Caprylic/CapricTriglyceride, Sodium Acrylates Copolymer C 1.0 Vitamin E AcetateTocopheryl Acetate 0.2 Bisabolol rac. Bisabolol QS Preservative QSPerfume D 3.0 Luviquat Care Polyquaternium-44 0.5 Luviquat Mono LSCocotrimonium Methosulfate 0.5 Cremophor A 25 Ceteareth-25 0.2D-Panthenol 50 P Panthenol, Propylene Glycol 4.0 1,2 Propylene GlycolPropylene Glycol Care 0.1 Edeta BD Disodium EDTA SA Compound QS Waterdem. Aqua dem.Production: Dissolve Phase A, then stir in Phase B. Fold in Phase C.Dissolve Phase D, stir it into the combined Phases A+B+C, homogenize andstir again for 15 min.Measure Values:

Viscosity: 7 200 mPa s Brookfield RVT

pH value: 5.8

The following formula provides an example of a daily care body sprayproduct with SPF 8 comprising the BBI-AV of the present invention.

DAILY CARE BODY SPRAY - SPF 8 % Ingredient INCI A 3.0 Uvinul MC 80Ethylhexyl Methoxycinnamate 2.0 Uvinul A Plus ™ DiethylaminoHydroxybenzoyl Hexyl Benzoate 1.0 Luviquat UltraCare Polyquaternium-443.0 1,2 Propylenglycol Care Propylene Glycol 2.0 D-Panthenol 50 PPanthenol, Propylene Glycol 1.0 Dow Corning 345 FluidCyclopentasiloxane, Cyclohexasiloxane 10.0  Eutanol G Octyldodecanol 0.5Luviskol K 30 PVP 10.0  Miglyol 812 Caprylic/Capric Triglyceride 3.0Finsolv TN C12-15 Alkyl Benzoate 3.0 Glycerin 87% Glycerin 1.0 Vitamin EAcetate Tocopheryl Acetate 0.3 Bisabolol rac. Bisabolol Compound QSEthanol AlcoholProduction: Weigh out the components of Phase A and dissolve themclearly.Measure Values:

SPF: 8 Colipa Task Force “Sun Protection Measurement”

The following formula provides an example of a daily care sun carelotion product with SPF 27 comprising the BBI-AV of the presentinvention.

SUN CARE LOTION - SPF 27 % Ingredient INCI A 4.5 Uvinul MC 80 EthylhexylMethoxycinnamate 2.0 Uvinul A Plus ™ Diethylamino Hydroxybenzoyl HexylBenzoate 3.0 Uvinul N 539 T Octocrylene 2.5 Cosmacol EMI Di-C12-13 AlkylMalate 0.5 Vitamin E Acetate Tocopheryl Acetate 4.0 Tego Care 450Polyglyceryl-3 Methyl Glucose Distearate B 3.5 Cetiol SN Deo CetearylIsononanoate 1.0 Ganex V-220 VP/Eicosene Copolymer 5.0 IsohexadecaneIsohexadecane 2.5 Cosmacol EMI Di-C12-13 Alkyl Malate 3.0 Uvinul TiO2Titanium Dioxide, Trimethoxycaprylylsilane C 5.0 Glycerin 87% Glycerin1.0 Lanette E Sodium Cetearyl Sulfate 0.5 Keltrol Xanthan Gum 60.7 Water dem. Aqua dem. D SA Compound 1.0 Phenonip Phenoxyethanol,Methylparaben, Ethylparaben, 0.3 Bisabolol rac. BisabololProduction: Heat Phases A and B separately to about 80° C. Stir Phase Binto Phase A whilst homogenizing. Heat Phase C to about 80° C. and stirit into the combined Phases A+B whilst homogenizing. Cool to about 40°C. add Phase D and homogenize again.Measure Values:

Viscosity: 3 200 mPa s Brookfield RVD II+

pH value: 6.0

SPF: 27 Colipa Task Force “Sun Protection Measurement”

SUN CARE LOTION - SPF 24 % Ingredient INCI A 2.0 Cremophor A 6Ceteareth-6, Stearyl Alcohol 2.0 Cremophor A 25 Ceteareth-25 3.0Syncrowax HRC Tribehenin 2.0 Lanette O Cetearyl Alcohol 2.0 Luvitol EHOCetearyl Ethylhexanoate 5.0 Uvinul MC 80 Ethylhexyl Methoxycinnamate 1.0Uvinul T 150 Ethylhexyl Triazone 1.0 Ganex V-220 VP/Eicosene Copolymer7.0 Isopropyl Myristate Isopropyl Myristate B 5.0 Z-Cote HP-1 ZincOxide, Triethoxycaprylylsilane C 0.2 Keltrol HydroxyethylAcrylate/Sodium Acryloyldimethyl Taurate Copolymer, Squalane,Polysorbate 60 0.2 Edeta BD Disodium EDTA 5.0 1,2 Propylene Glycol CarePropylene Glycol 0.5 D-Panthenol USP Panthenol 61.9  Water dem. Aquadem. D SA Compound 0.5 Euxyl K 300 Phenoxyethanol, Methylparaben,Butylparaben, Ethylparaben, Propylparaben, Isobutylparaben 1.0 Vitamin EAcetate Tocopheryl Acetate 0.2 Bisabolol rac. BisabololProduction: Heat Phase A to 80° C., add Phase B and homogenize for 3min. Heat Phase C to about 80° C., and stir it into the combined PhasesA+B whilst homogenizing. Cool to about 40° C., add Phase D, andhomogenize.Measure Values:

Viscosity: 5 000 mPa s Brookfield RVD II+

pH value: 7.5

SPF: 24 Colipa Task Force “Sun Protection Measurement”

The following formula provides an example of a sun screen emulsionproduct with SPF 28 comprising the BBI-AV of the present invention.

SUN SCREEN EMULSION - SPF 28 % Ingredient INCI A 3.5 Cremophor A 6Ceteareth-6, Stearyl Alcohol 1.5 Cremophor A 25 Ceteareth-25 7.5 UvinulMC 80 Ethylhexyl Methoxycinnamate 2.0 Uvinul A Plus ™ DiethylaminoHydroxybenzoyl Hexyl Benzoate 2.0 Dow Corning 345 FluidCyclopentasiloxane, Cyclohexasiloxane 0.5 Bees Wax 3044 PH Bees Wax 3.0Lanette O Cetearyl Alcohol 10.0  Miglyol 812 Caprylic/CapricTriglyceride B 5.0 T-Lite SF-S Titanium Dioxide, Silica, Methicone,Alumina C 3.0 Glycerin 87% Glycerin 0.2 Edeta BD Disodium EDTA 0.3Keltrol T Xanthan Gum 1.0 Plantacare 2000 Decyl Glucoside 2.0D-Panthenol 50 P Panthenol, Propylene Glycol 57.3  Water dem. Aqua dem.D SA Compound 1.0 Vitamin E Acetate Tocopheryl Acetate 0.2 Bisabololrac. Bisabolol QS Perfume QS PreservativeProduction: Heat Phase A to 80° C., add Phase B and homogenize for 3min. Heat Phase C to about 80° C., and stir it into the combined PhasesA+B whilst homogenizing. Cool to about 40° C., add Phase D andhomogenize.Measure Values:

Viscosity: 7 500 mPa s Brookfield RVD II+

pH value: 6.6

SPF: 28 Colipa Task Force “Sun Protection Measurement”

The following formula provides an example of a foot balm productcomprising the BBI-AV of the present invention.

FOOT BALM % Ingredient INCI A 2.0 Cremophor A 6 Ceteareth-6, StearylAlcohol 2.0 Cremophor A 25 Ceteareth-25 5.0 Luvitol EHO CetearylEthylhexanoate 4.0 Lanette 16 Cetyl Alcohol 4.0 Cutina Gms GlycerylStearate 5.0 Paraffin Oil Mineral Oil 0.2 Menthol Menthol 0.5 CamphorCamphor B 70.3  Water dem. Aqua dem. QS Preservative C SA Compound 1.0Bisabolol rac. Bisabolol 1.0 Vitamin E Acetate Tocopheryl Acetate D 5.0Witch Hazel Extract Witch Hazel ExtractProduction: Heat Phases A and B to about 80° C. separately. Stir Phase Binto Phase A whilst homogenizing. Cool to about 40° C., add Phases C andD and homogenize again. Cool to room temperature.Measure Values:

Viscosity: 20 500 mPa s Brookfield RVD II+

pH value: 6.0

The following formula provides an example of a refreshing foot gelproduct comprising the BBI-AV of the present invention.

REFRESHING FOOT GEL % Ingredient INCI A 0.6 Carbopol Ultrez 21Acrylates/C10-30 Alkyl Acrylate Crosspolymer 45.9  Water dem. Aqua dem.B 1.0 Bisabolol rac. Bisabolol 0.5 Farnesol Farnesol q.s. Perfume 4.5Cremophor CO 40 PEG-40 Hydrogenated Castor Oil 1.0 Neutrol TETetrahydroxypropyl Ethylenediamine 1.5 Menthol Menthol SA Compound 45.0 Ethanol 96% Alcohol QS FD&C Blue No. 1 C.I. 42 090, FD&C Blue No. 1Production: Phase A: Intersperse the Carbopol and let it settle on thebottom of the beaker. Dissolve Phase B and stir it into Phase A.Measure Values:

Viscosity: 14 500 mPa s Brookfield RVD II+

pH value: 7.5

The following formula provides an example of a skin conditioning gelproduct comprising the BBI-AV of the present invention.

Skin Conditioning Gel % Ingredient INCI A 3.6 Cremophor CO 40 PEG-40Hydrogenated Castor Oil 15.0  Ethanol Alcohol 0.1 Bisabolol rac.Bisabolol 0.5 Vitamin E Acetate Tocopheryl Acetate QS Perfume B 3.0D-Panthenol USP Panthenol 0.6 Carbopol 940 Carbomer SA Compound 76.4 Water dem. Aqua dem. C 0.8 Triethanolamine Care TriethanolamineProduction: Dissolve Phase A clearly. Allow Phase B to swell andneutralize with Phase C. Stir Phase A into the neutralized Phase B andhomogenize.Measure Values:

Viscosity: 57 600 mPa s Brookfield RVD II+

pH value: 7.7

The following formula provides an example of a W/O emulsion comprisingthe BBI-AV of the present invention.

W/O EMULSION % Ingredient INCI A 6.0 Cremophor WO 7 PEG-7 HydrogenatedCastor Oil 8.0 Luvitol EHO Cetearyl Ethylhexanoate 5.0 IsopropylMyristate Isopropyl Myristate 15.0  Paraffin Oil Mineral Oil 0.3Magnesium Stearate Magnesium Stearate 0.3 Aluminum Stearate AluminumStearate 2.0 Elfacos ST9 PEG-45/Dodecyl Glycol Copolymer B 5.0 Glycerin87% Glycerin 0.7 Magnesium Magnesium Sulfate Sulfate-7-hydrate 56.6 Water dem. Aqua dem. C SA Compound 0.5 Vitamin E Acetate TocopherylAcetate 0.6 Bisabolol rac. BisabololProduction: Heat Phases A and B separately to about 85° C. Stir Phase Binto Phase

A and homogenize. Cool to about 400C whilst stirring, add Phase C andhomogenize again. Cool to room temperature.

Measure Values:

Viscosity: 37 500 mPa s Brookfield RVD II+

The following formula provides an example of a O/W emulsion productcomprising the BBI-AV of the present invention.

O/W EMULSION % Ingredient INCI A 1.7 Cremophor A 6 Ceteareth-6, StearylAlcohol 0.7 Cremophor A 25 Ceteareth-25 2.0 Uvinul A Plus ™ DiethylaminoHydroxybenzoyl Hexyl Benzoate 2.0 Abil B 8843 PEG-14 Dimethicone 3.6Lanette O Cetearyl Alcohol 6.0 Uvinul MC 80 Ethylhexyl Methoxycinnamate2.0 Cetiol B Dibutyl Adipate B 5.0 Glycerin 87% Glycerin 0.2 Edeta BDDisodium EDTA 1.0 D-Panthenol 75 W Panthenol q.s. Preservative 68.8 Water dem. Aqua dem. C 4.0 Luvigel EM Caprylic/Capric Triglyceride,Sodium Acrylates Copolymer D 0.2 Sodium Ascorbyl Sodium AscorbylPhosphate Phosphate 1.0 Vitamin E Acetate Tocopheryl Acetate 0.2Bisabolol rac. Bisabolol E q.s. Sodium Hydroxide Sodium Hydroxide 10%aq. w/w F 1.0 RetiSTAR Caprylic/Capric Triglyceride, Sodium Ascorbate,Tocopherol, Retinol SA CompoundProduction: Heat Phase A and B separately to about 80° C. Stir Phase Binto Phase A and homogenize. Stir Phase C into the combined Phases A+Band homogenize. Cool to about 40° C., add Phase D, then adjust the pHvalue with Phase E to 6.5. Add Phase F and homogenize. Cool to roomtemperature.Measure Values:

Viscosity: 37 500 mPa s Brookfield RVD II+

pH value: 6.3

The following formula provides an example of a protective day creamproduct comprising the BBI-AV of the present invention.

PROTECTIVE DAY CREAM % Ingredient INCI A 1.7 Cremophor A 6 Ceteareth-6,Stearyl Alcohol 0.7 Cremophor A 25 Ceteareth-25 2.0 Uvinul A Plus ™Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Abil B 8843 PEG-14Dimethicone 3.6 Lanette O Cetearyl Alcohol 6.0 Uvinul MC 80 EthylhexylMethoxycinnamate 2.0 Cetiol B Dibutyl Adipate B 5.0 Glycerin 87%Glycerin 0.2 Edeta BD Disodium EDTA 1.0 D-Panthenol 75 W Panthenol QSPreservative 69.6  Water dem. Aqua dem. C 4.0 Luvigel EM Caprylic/CapricTriglyceride, Sodium Acrylates Copolymer D 1.0 Sodium Ascorbyl SodiumAscorbyl Phosphate Phosphate 1.0 Vitamin E Acetate Tocopheryl Acetate SACompound 0.2 Bisabolol rac. Bisabolol E QS Sodium Hydroxide SodiumHydroxide 10% aq. w/wProduction: Heat Phase A and B separately to about 80° C. Stir Phase Binto Phase A and homogenize. Stir Phase C into the combined Phases A+Band homogenize. Cool to about 40° C., add Phase D, then adjust the pHvalue with Phase E to 6.5 and homogenize. Cool to room temperature.Measure Values:

Viscosity: 24 000 mPa s Brookfield RVD II+

pH value: 6.4

The following formulae provide examples of hair care products comprisingthe BBI-AV of the present invention.

HAIR CONDITIONERS % Ingredients (INCI) A 10.0  PVP/VA Copolymer 0.2Hydroxyethyl Cetyldimonium Phosphate 0.2 Ceteareth-25 0.5 DimethiconeCopolyol QS Perfume 10.0  Alcohol SA Compound 68.1  Aqua dem. B 10.0 Propane/Butane A 10.0  PVP/VA Copolymer 0.2 Hydroxyethyl CetyldimoniumPhosphate 0.2 Ceteareth-25 0.5 Dimethicone Copolyol QS Perfume 10.0 Alcohol SA Compound 64.1  Aqua dem. B 10.0  Propane/ButaneProduction: Add all compounds to Phase A and stir to homogenize. Fillinto appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-4 0.5Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-4 0.5Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative87.5  Aqua dem. B 6.0 Propane/ButaneProduction: Add all compounds to Phase A and stir to homogenize. Fillinto appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-11 0.5Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-11 0.5Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QSPreservative 87.5  Aqua dem. B 6.0 Propane/ButaneProduction: Add all compounds to Phase A and stir to homogenize. Fillinto appropriate container and charge with Phase B.

STYLING FOAMS % Ingredients (INCI) A 0.5 Laureth-4 QS Perfume B 77.3 Aqua dem. 10.0  Polyquaternium-28 Compound 0.5 Dimethicone Copolyol 0.2Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C10.0  HFC 152 A A 0.5 Laureth-4 QS Perfume B 73.3  Aqua dem. 10.0 Polyquaternium-28 SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-250.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152AProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Fill into appropriatecontainer and charge with Phase C.

% Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QS Perfume B 78.5 Aqua dem. 6.7 Acrylates Copolymer 0.6 AMP SA Compound 0.5 DimethiconeCopolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2Hydroxyethylcellulose C 10.0  HFC 152 A A 2.0 Cocotrimonium MethosulfateQS Perfume B 74.5  Aqua dem. 6.7 Acrylates Copolymer 0.6 AMP SA Compound0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA0.2 Hydroxyethylcellulose C 10.0  HFC 152 AProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Fill into appropriatecontainer and charge with Phase C.

STYLING FOAM % Ingredients (INCI) A  2.0 Cocotrimonium Methosulfate QSPerfume B  7.70 Polyquaternium-44 SA Compound QS Preservative 79.3 Aquadem. C 10.0 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

STYLING FOAM % Ingredients (INCI) A 2.00 Cocotrimonium Methosulfate QSPerfume B 72.32  Aqua dem. 2.00 VP/Acrylates/Lauryl MethacrylateCopolymer 0.53 AMP SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-1215.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00Cocotrimonium Methosulfate QS Perfume B 68.32  Aqua dem. 2.00VP/Acrylates/Lauryl Methacrylate Copolymer 0.53 AMP SA Compound 0.20Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone,Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20Hydroxyethylcellulose D 6.00 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Add Phase C and homogenizeagain. Adjust pH to 6-7. Fill into appropriate container and charge withPhase D.

STYLING FOAMS % Ingredients (INCI) A 2.00 Cetrimonium Chloride QSPerfume B 67.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone,Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00 Cetrimonium ChlorideQS Perfume B 63.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone,Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20Hydroxyethylcellulose D 6.00 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Add Phase C and homogenizeagain. Adjust pH to 6-7. Fill into appropriate container and charge withPhase D.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor OilQS Perfume 85.5  Aqua dem. B 7.0 Sodium Polystyrene Sulfonate SACompound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane AQS PEG-40 Hydrogenated Castor Oil QS Perfume 81.5  Aqua dem. B 7.0Sodium Polystyrene Sulfonate SA Compound 0.5 Cetrimonium Bromide QSPreservative C 6.0 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor OilQS Perfume 92.0  Aqua dem. B 0.5 Polyquaternium-10 1.0 Compound 0.5Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane A QS PEG-40Hydrogenated Castor Oil QS Perfume 88.0  Aqua dem. B 0.5Polyquaternium-10 5.0 Compound 0.5 Cetrimonium Bromide QS Preservative C6.0 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

% Ingredients (INCI) A QS PEG-40 Hydrogenated Castor Oil QS Perfume82.5  Aqua dem. B 10.0  Polyquaternium-16 SA Compound 0.5 HydroxyethylCetyldimonium Phosphate QS Preservative C 6.0 Propane/Butane A QS PEG-40Hydrogenated Castor Oil QS Perfume 78.5  Aqua dem. B 100   Polyquaternium-16 SA Compound 0.5 Hydroxyethyl Cetyldimonium PhosphateQS Preservative C 6.0 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QSPerfume B 84.0  Aqua dem. 2.0 Chitosan SA Compound 0.5 DimethiconeCopolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152A A 2.0 Cocotrimonium Methosulfate QS Perfume B 80.0  Aqua dem. 2.0Chitosan SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152 AProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

SHAMPOOS % Ingredients (INCI) A 30.0  Sodium Laureth Sulfate 6.0 SodiumCocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0 Sodium Laureth Sulfate,Glycol Distearate, Cocamide MEA, Laureth-10 SA Compound 7.7Polyquaternium-44 2.0 Amodimethicone QS Perfume QS Preservative 1.0Sodium Chloride 43.3  Aqua dem. B QS Citric Acid A 30.0  Sodium LaurethSulfate 6.0 Sodium Cocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0Sodium Laureth Sulfate, Glycol Distearate, Cocamide MEA, Laureth-10Compound 7.7 Polyquaternium-44 2.0 Amodimethicone QS Perfume QSPreservative 1.0 Sodium Chloride 39.3  Aqua dem. B QS Citric AcidProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7 with citric acid.

SHOWER GELS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0Decyl Glucoside 5.0 Cocamidopropyl Betaine SA Compound 1.0 Panthenol QSPerfume QS Preservative 2.0 Sodium Chloride 46.0  Aqua dem. B QS CitricAcid A 40.0  Sodium Laureth Sulfate 5.0 Decyl Glucoside 5.0Cocamidopropyl Betaine SA Compound 1.0 Panthenol QS Perfume QSPreservative 2.0 Sodium Chloride 42.0  Aqua dem. B QS Citric AcidProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0 SodiumC12-15 Pareth-15 Sulfonate 5.0 Decyl Glucoside QS Perfume 0.1Phytantriol 44.6  Aqua dem. SA Compound 0.3 Polyquaternium-10 1.0Panthenol QS Preservative 1.0 Laureth-3 2.0 Sodium Chloride A 40.0 Sodium Laureth Sulfate 5.0 Sodium C12-15 Pareth-15 Sulfonate 5.0 DecylGlucoside QS Perfume 0.1 Phytantriol 40.6  Aqua dem. SA Compound 0.3Polyquaternium-10 1.0 Panthenol QS Preservative 1.0 Laureth-3 2.0 SodiumChlorideProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 15.00  Cocamidopropyl Betaine 10.00 Disodium Cocoamphodiacetate 5.00 Polysorbate 20 5.00 Decyl Glucoside QSPerfume QS Preservative SA Compound 0.15 Guar HydroxypropyltrimoniumChloride 2.00 Laureth-3 58.00  Aqua dem. QS Citric Acid B 3.00 PEG-150Distearate A 15.00  Cocamidopropyl Betaine 10.00  DisodiumCocoamphodiacetate 5.00 Polysorbate 20 5.00 Decyl Glucoside QS PerfumeQS Preservative Compound 0.15 Guar Hydroxypropyltrimonium Chloride 2.00Laureth-3 54.00  Aqua dem. QS Citric Acid B 3.00 PEG-150 DistearateProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7. Add Phase B and heat to max. 40° C.

BODY LOTIONS % Ingredients (INCI) A 2.0 Ceteareth-25 2.0 Ceteareth-6,Stearyl Alcohol 3.0 Cetearyl Ethylhexanoate 1.0 Dimethicone 4.0 CetearylAlcohol 3.0 Glyceryl Stearate SE 5.0 Mineral Oil 4.0 SimmondsiaChinensis (Jojoba) Seed Oil 3.0 Mineral Oil, Lanolin Alcohol B 5.0Propylene Glycol SA Compound 1.0 Panthenol 0.5 Magnesium AluminumSilicate QS Preservative 65.5  Aqua dem. C QS Perfume D QS Citric Acid A2.0 Ceteareth-25 2.0 Ceteareth-6, Stearyl Alcohol 3.0 CetearylEthylhexanoate 1.0 Dimethicone 4.0 Cetearyl Alcohol 3.0 GlycerylStearate SE 5.0 Mineral Oil 4.0 Simmondsia Chinensis (Jojoba) Seed Oil3.0 Mineral Oil, Lanolin Alcohol B 5.0 Propylene Glycol SA Compound 1.0Panthenol 0.5 Magnesium Aluminum Silicate QS Preservative 61.5  Aquadem. C QS Perfume D QS Citric AcidProduction: Heat Phases A and B separately to approx. 40° C. Add Phase Bto Phase A and homogenize by stirring. Add Phase C to the combined PhaseA and B and homogenize again. Adjust pH with Phase D to 6-7. Homogenizeby stirring and cool to room temperature.

BODY LOTIONS % Ingredients (INCI) A 6.0 PEG-7 Hydrogenated Castor Oil10.0  Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil0.5 Shea Butter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5Magnesium Stearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0Dipropylene Glycol 0.7 Magnesium Sulfate QS Preservative 62.9  Aqua dem.C QS Perfume SA Compound A 6.0 PEG-7 Hydrogenated Castor Oil 10.0 Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil 0.5 SheaButter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5 MagnesiumStearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0 DipropyleneGlycol 0.7 Magnesium Sulfate QS Preservative 58.9  Aqua dem. C QSPerfume SA CompoundProduction: Heat Phases A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Cool to 40° C. and add Phase C.Homogenize again and cool to room temperature.

HAIR CONDITIONERS % Ingredients (INCI) A 10.0  PVP/VA Copolymer 0.2Hydroxyethyl Cetyldimonium Phosphate 0.2 Ceteareth-25 0.5 DimethiconeCopolyol QS Perfume 10.0  Alcohol SA Compound 68.1  Aqua dem. B 10.0 Propane/Butane A 10.0  PVP/VA Copolymer 0.2 Hydroxyethyl CetyldimoniumPhosphate 0.2 Ceteareth-25 0.5 Dimethicone Copolyol QS Perfume 10.0 Alcohol SA Compound 64.1  Aqua dem. B 10.0  Propane/ButaneProduction: Add all compounds to Phase A and stir to homogenize. Fillinto appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-4 0.5Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QSPreservative 91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-40.5 Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QSPreservative 87.5  Aqua dem. B 6.0 Propane/ButaneProduction: Add all compounds to Phase A and stir to homogenize. Fillinto appropriate container and charge with Phase B.

FOAM CONDITIONERS % Ingredients (INCI) A 1.0 Polyquaternium-11 0.5Hydroxyethyl Cetyldimonium Phosphate Compound QS Perfume QS Preservative91.5  Aqua dem. B 6.0 Propane/Butane A 1.0 Polyquaternium-11 0.5Hydroxyethyl Cetyldimonium Phosphate SA Compound QS Perfume QSPreservative 87.5  Aqua dem. B 6.0 Propane/ButaneProduction: Add all compounds to Phase A and stir to homogenize. Fillinto appropriate container and charge with Phase B.

STYLING FOAMS % Ingredients (INCI) A 0.5 Laureth-4 QS Perfume B 77.3 Aqua dem. 10.0  Polyquaternium-28 SA Compound 0.5 Dimethicone Copolyol0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA 0.2 HydroxyethylcelluloseC 10.0  HFC 152 A A 0.5 Laureth-4 QS Perfume B 73.3  Aqua dem. 10.0 Polyquaternium-28 SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-250.2 Panthenol 0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152AProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Fill into appropriatecontainer and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QSPerfume B 78.5  Aqua dem. 6.7 Acrylates Copolymer 0.6 AMP SA Compound0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA0.2 Hydroxyethylcellulose C 10.0  HFC 152 A A 2.0 CocotrimoniumMethosulfate QS Perfume B 74.5  Aqua dem. 6.7 Acrylates Copolymer 0.6AMP SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2 Panthenol0.1 PEG-25 PABA 0.2 Hydroxyethylcellulose C 10.0  HFC 152 AProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Fill into appropriatecontainer and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QSPerfume B  7.70 Polyquaternium-44 SA Compound QS Preservative 79.3  Aquadem. C 10.0  Propane/Butane A 2.0 Cocotrimonium Methosulfate QS PerfumeB  7.70 Polyquaternium-44 SA Compound QS Preservative 75.3  Aqua dem. C10.0  Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.00 Cocotrimonium Methosulfate QSPerfume B 72.32  Aqua dem. 2.00 VP/Acrylates/Lauryl MethacrylateCopolymer 0.53 AMP SA Compound 0.20 Ceteareth-25 0.50 Panthenol 0.05Benzophenone-4 0.20 Amodimethicone, Cetrimonium Chloride, Trideceth-1215.00  Alcohol C 0.20 Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00Cocotrimonium Methosulfate QS Perfume B 68.32  Aqua dem. 2.00VP/Acrylates/Lauryl Methacrylate Copolymer 0.53 AMP SA Compound 0.20Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone,Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20Hydroxyethylcellulose D 6.00 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Add Phase C and homogenizeagain. Adjust pH to 6-7. Fill into appropriate container and charge withPhase D.

STYLING FOAMS % Ingredients (INCI) A 2.00 Cetrimonium Chloride QSPerfume B 67.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone,Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20Hydroxyethylcellulose D 6.00 Propane/Butane A 2.00 Cetrimonium ChlorideQS Perfume B 63.85  Aqua dem. 7.00 Polyquaternium-46 SA Compound 0.20Ceteareth-25 0.50 Panthenol 0.05 Benzophenone-4 0.20 Amodimethicone,Cetrimonium Chloride, Trideceth-12 15.00  Alcohol C 0.20Hydroxyethylcellulose D 6.00 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Add Phase C and homogenizeagain. Adjust pH to 6-7. Fill into appropriate container and charge withPhase D.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor OilQS Perfume 85.5  Aqua dem. B 7.0 Sodium Polystyrene Sulfonate SACompound 0.5 Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane AQS PEG-40 Hydrogenated Castor Oil QS Perfume 81.5  Aqua dem. B 7.0Sodium Polystyrene Sulfonate SA Compound 0.5 Cetrimonium Bromide QSPreservative C 6.0 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor OilQS Perfume 92.0  Aqua dem. B 0.5 Polyquaternium-10 1.0 Compound 0.5Cetrimonium Bromide QS Preservative C 6.0 Propane/Butane A QS PEG-40Hydrogenated Castor Oil QS Perfume 88.0  Aqua dem. B 0.5Polyquaternium-10 5.0 Compound 0.5 Cetrimonium Bromide QS Preservative C6.0 Propane/ButaneProduction: Weigh out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A QS PEG-40 Hydrogenated Castor OilQS Perfume 82.5  Aqua dem. B 10.0  Polyquaternium-16 SA Compound 0.5Hydroxyethyl Cetyldimonium Phosphate QS. Preservative C 6.0Propane/Butane A QS PEG-40 Hydrogenated Castor Oil QS Perfume 78.5  Aquadem. B 10.0  Polyquaternium-16 SA Compound 0.5 HydroxyethylCetyldimonium Phosphate QS Preservative C 6.0 Propane/ButaneProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

STYLING FOAMS % Ingredients (INCI) A 2.0 Cocotrimonium Methosulfate QSPerfume B 84.0  Aqua dem. 2.0 Chitosan SA Compound 0.5 DimethiconeCopolyol 0.2 Ceteareth-25 0.2 Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152A A 2.0 Cocotrimonium Methosulfate QS Perfume B 80.0  Aqua dem. 2.0Chitosan SA Compound 0.5 Dimethicone Copolyol 0.2 Ceteareth-25 0.2Panthenol 0.1 PEG-25 PABA C 10.0  HFC 152 AProduction: Weight out the compounds of Phase A and mix them. DissolvePhase B, stir into Phase A and homogenize. Adjust pH to 6-7. Fill intoappropriate container and charge with Phase C.

SHAMPOOS % Ingredients (INCI) A 30.0  Sodium Laureth Sulfate 6.0 SodiumCocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0 Sodium Laureth Sulfate,Glycol Distearate, Cocamide MEA, Laureth-10 SA Compound 7.7Polyquaternium-44 2.0 Amodimethicone QS Perfume QS Preservative 1.0Sodium Chloride 43.3  Aqua dem. B QS Citric Acid A 30.0  Sodium LaurethSulfate 6.0 Sodium Cocoamphoacetate 6.0 Cocamidopropyl Betaine 3.0Sodium Laureth Sulfate, Glycol Distearate, Cocamide MEA, Laureth-10 SACompound 7.7 Polyquaternium-44 2.0 Amodimethicone SA Perfume SA.Preservative 1.0 Sodium Chloride 39.3  Aqua dem. B QS Citric AcidProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7 with citric acid.

SHOWER GELS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0Decyl Glucoside 5.0 Cocamidopropyl Betaine SA Compound 1.0 Panthenol QSPerfume QS Preservative 2.0 Sodium Chloride 46.0  Aqua dem. B QS CitricAcid A 40.0  Sodium Laureth Sulfate 5.0 Decyl Glucoside 5.0Cocamidopropyl Betaine SA Compound 1.0 Panthenol QS Perfume QSPreservative 2.0 Sodium Chloride 42.0  Aqua dem. B QS Citric AcidProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 40.0  Sodium Laureth Sulfate 5.0 SodiumC12-15 Pareth-15 Sulfonate 5.0 Decyl Glucoside QS Perfume 0.1Phytantriol 44.6  Aqua dem. SA Compound 0.3 Polyquaternium-10 1.0Panthenol QS Preservative 1.0 Laureth-3 2.0 Sodium Chloride A 40.0 Sodium Laureth Sulfate 5.0 Sodium C12-15 Pareth-15 Sulfonate 5.0 DecylGlucoside QS Perfume 0.1 Phytantriol 40.6  Aqua dem. SA Compound 0.3Polyquaternium-10 1.0 Panthenol QS Preservative 1.0 Laureth-3 2.0 SodiumChlorideProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7 with citric acid.

SHAMPOOS % Ingredients (INCI) A 15.00 Cocamidopropyl Betaine 10.00Disodium Cocoamphodiacetate  5.00 Polysorbate 20  5.00 Decyl GlucosideQS Perfume QS Preservative SA Compound  0.15 Guar HydroxypropyltrimoniumChloride  2.00 Laureth-3 58.00 Aqua dem. QS Citric Acid B  3.00 PEG-150Distearate A 15.00 Cocamidopropyl Betaine 10.00 DisodiumCocoamphodiacetate  5.00 Polysorbate 20  5.00 Decyl Glucoside QS PerfumeQS Preservative SA Compound  0.15 Guar Hydroxypropyltrimonium Chloride 2.00 Laureth-3 54.00 Aqua dem. QS Citric Acid B  3.00 PEG-150DistearateProduction: Weight out the compounds of Phase A and mix them. Adjust pHto 6-7. Add Phase B and heat to max. 40° C.

BODY LOTIONS % Ingredients (INCI) A 2.0 Ceteareth-25 2.0 Ceteareth-6,Stearyl Alcohol 3.0 Cetearyl Ethylhexanoate 1.0 Dimethicone 4.0 CetearylAlcohol 3.0 Glyceryl Stearate SE 5.0 Mineral Oil 4.0 SimmondsiaChinensis (Jojoba) Seed Oil 3.0 Mineral Oil, Lanolin Alcohol B 5.0Propylene Glycol SA Compound 1.0 Panthenol 0.5 Magnesium AluminumSilicate QS Preservative 65.5  Aqua dem. C QS Perfume D QS Citric Acid A2.0 Ceteareth-25 2.0 Ceteareth-6, Stearyl Alcohol 3.0 CetearylEthylhexanoate 1.0 Dimethicone 4.0 Cetearyl Alcohol 3.0 GlycerylStearate SE 5.0 Mineral Oil 4.0 Simmondsia Chinensis (Jojoba) Seed Oil3.0 Mineral Oil, Lanolin Alcohol B 5.0 Propylene Glycol SA Compound 1.0Panthenol 0.5 Magnesium Aluminum Silicate QS Preservative 61.5  Aquadem. C QS Perfume D QS Citric AcidProduction: Heat Phases A and B separately to approx. 40° C. Add Phase Bto Phase A and homogenize by stirring. Add Phase C to the combined PhaseA and B and homogenize again. Adjust pH with Phase D to 6-7. Homogenizeby stirring and cool to room temperature.

BODY LOTIONS % Ingredients (INCI) A 6.0 PEG-7 Hydrogenated Castor Oil10.0  Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil0.5 Shea Butter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5Magnesium Stearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0Dipropylene Glycol 0.7 Magnesium Sulfate QS Preservative 62.9  Aqua dem.C QS. Perfume SA Compound A 6.0 PEG-7 Hydrogenated Castor Oil 10.0 Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 7.0 Mineral Oil 0.5 SheaButter (Butyrospermum Parkii) 0.5 Aluminum Stearate 0.5 MagnesiumStearate 0.2 Bisabolol 0.7 Quaternium-18-Hectorite B 5.0 DipropyleneGlycol 0.7 Magnesium Sulfate QS Preservative 58.9  Aqua dem. C QSPerfume SA CompoundProduction: Heat Phases A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Cool to 40° C. and add Phase C.Homogenize again and cool to room temperature.

DAILY SKIN CARE O/W % Ingredients (INCI) A 1.7 Ceteareth-6, StearylAlcohol 0.7 Ceteareth-25 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate2.0 PEG-14 Dimethicone 3.6 Cetearyl Alcohol 6.0 EthylhexylMethoxycinnamate 2.0 Dibutyl Adipate B 5.0 Glycerin 0.2 Disodium EDTA1.0 Panthenol QS Preservative 67.8  Aqua dem. C 4.0 Caprylic/CapricTriglyceride, Sodium Acrylates Copolymer D 0.2 Sodium Ascorbyl Phosphate1.0 Tocopheryl Acetate 0.2 Bisabolol 1.0 Caprylic/Capric Triglyceride,Sodium Ascorbate, Tocopherol, Retinol SA Compound E QS Sodium HydroxideA 1.7 Ceteareth-6, Stearyl Alcohol 0.7 Ceteareth-25 2.0 DiethylaminoHydroxybenzoyl Hexyl Benzoate 2.0 PEG-14 Dimethicone 3.6 CetearylAlcohol 6.0 Ethylhexyl Methoxycinnamate 2.0 Dibutyl Adipate B 5.0Glycerin 0.2 Disodium EDTA 1.0 Panthenol QS Preservative 63.8  Aqua dem.C 4.0 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 0.2Sodium Ascorbyl Phosphate 1.0 Tocopheryl Acetate 0.2 Bisabolol 1.0Caprylic/Capric Triglyceride, Sodium Ascorbate, Tocopherol, Retinol SACompound E QS Sodium HydroxideProduction: Heat Phase A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Add Phase C to the combined PhaseA and B and homogenize again. Cool to approx. 40° C. and add Phase D.Adjust pH with Phase E to approx. 6.5. Homogenize by stirring and coolto room temperature.

PROTECTIVE DAY SKIN CREME O/W % Ingredients (INCI) A 1.7 Ceteareth-6,Stearyl Alcohol 0.7 Ceteareth-25 2.0 Diethylamino Hydroxybenzoyl HexylBenzoate 2.0 PEG-14 Dimethicone 3.6 Cetearyl Alcohol 6.0 EthylhexylMethoxycinnamate 2.0 Dibutyl Adipate B 5.0 Glycerin 0.2 Disodium EDTA1.0 Panthenol QS Preservative 68.6  Aqua dem. C 4.0 Caprylic/CapricTriglyceride, Sodium Acrylates Copolymer D 1.0 Sodium Ascorbyl Phosphate1.0 Tocopheryl Acetate 0.2 Bisabolol SA Compound E QS Sodium Hydroxide A1.7 Ceteareth-6, Stearyl Alcohol 0.7 Ceteareth-25 2.0 DiethylaminoHydroxybenzoyl Hexyl Benzoate 2.0 PEG-14 Dimethicone 3.6 CetearylAlcohol 6.0 Ethylhexyl Methoxycinnamate 2.0 Dibutyl Adipate B 5.0Glycerin 0.2 Disodium EDTA 1.0 Panthenol QS Preservative 64.6  Aqua dem.C 4.0 Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer D 1.0Sodium Ascorbyl Phosphate 1.0 Tocopheryl Acetate 0.2 Bisabolol CompoundE QS Sodium HydroxideProduction: Heat Phase A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Add Phase C to the combined PhaseA and B and homogenize again. Cool to approx. 40° C. and add Phase D.Adjust pH with Phase E to approx. 6.5. Homogenize by stirring and coolto room temperature.

FACIAL CLEANSER O/W % Ingredients (INCI) A 10.0  Cetearyl Ethylhexanoate10.0  Caprylic/Capric Triglyceride 1.5 Cyclopentasiloxane,Cyclohexasilosane 2.0 PEG-40 Hydrogenated Castor Oil B 3.5Caprylic/Capric Triglyceride, Sodium Acrylates Copolymer C 1.0Tocopheryl Acetate 0.2 Bisabolol QS Preservative QS Perfume D 3.0Polyquaternium-44 0.5 Cocotrimonium Methosulfate 0.5 Ceteareth-25 2.0Panthenol, Propylene Glycol 4.0 Propylene Glycol 0.1 Disodium EDTA SACompound 60.7  Aqua dem. A 10.0  Cetearyl Ethylhexanoate 10.0 Caprylic/Capric Triglyceride 1.5 Cyclopentasiloxane, Cyclohexasilosane2.0 PEG-40 Hydrogenated Castor Oil B 3.5 Caprylic/Capric Triglyceride,Sodium Acrylates Copolymer C 1.0 Tocopheryl Acetate 0.2 Bisabolol QSPreservative QS Perfume D 3.0 Polyquaternium-44 0.5 CocotrimoniumMethosulfate 0.5 Ceteareth-25 2.0 Panthenol, Propylene Glycol 4.0Propylene Glycol 0.1 Disodium EDTA SA Compound 56.7  Aqua dem.Production: Dissolve Phase A and add Phase B to Phase A and homogenizeby stirring. Add. Phase C to the combined Phase A and B and homogenizeagain. Add. Phase D to the combined Phase A, B and C and homogenizeagain. Dissolve Phase D and add to Phase A, B, and C and homogenizeagain. Stir for 15 minutes.

DAILY CARE BODY SPRAY % Ingredients (INCI) A 3.0 EthylhexylMethoxycinnamate 2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 1.0Polyquaternium-44 3.0 Propylene Glycol 2.0 Panthenol, Propylene Glycol1.0 Cyclopentasiloxane, Cyclohexasiloxane 10.0  Octyldodecanol 0.5 PVP10.0  Caprylic/Capric Triglyceride 3.0 C12-15 Alkyl Benzoate 3.0Glycerin 1.0 Tocopheryl Acetate 0.3 Bisabolol SA Compound 59.2  AlcoholA 3.0 Ethylhexyl Methoxycinnamate 2.0 Diethylamino Hydroxybenzoyl HexylBenzoate 1.0 Polyquaternium-44 3.0 Propylene Glycol 2.0 Panthenol,Propylene Glycol 1.0 Cyclopentasiloxane, Cyclohexasiloxane 10.0 Octyldodecanol 0.5 PVP 10.0  Caprylic/Capric Triglyceride 3.0 C12-15Alkyl Benzoate 3.0 Glycerin 1.0 Tocopheryl Acetate 0.3 Bisabolol SACompound 55.2  AlcoholProduction: Weight all ingredients of Phase A and dissolve completely bystirring.

SKIN CARE GEL % Ingredients (INCI) A 3.6 PEG-40 Hydrogenated Castor Oil15.0 Alcohol 0.1 Bisabolol 0.5 Tocopheryl Acetate QS Perfume B 3.0Panthenol 0.6 Carbomer SA Compound 75.4 Aqua dem, C 0.8 TriethanolamineA 3.6 PEG-40 Hydrogenated Castor Oil 15.0 Alcohol 0.1 Bisabolol 0.5Tocopheryl Acetate QS Perfume B 3.0 Panthenol 0.6 Carbomer SA Compound71.4 Aqua dem, C 0.8 TriethanolamineProduction: Dissolve Phase A. Swell Phase B and neutralize with Phase C.Add Phase A to Phase B and C and homogenize by stirring.

AFTER SHAVE LOTIONS % Ingredients (INCI) A 10.0 Cetearyl Ethylhexanoate5.0 Tocopheryl Acetate 1.0 Bisabolol 0.1 Perfume 0.3 Acrylates/C10-30Alkyl Acrylate Crosspolymer B 15.0 Alcohol 1.0 Panthenol 3.0 Glycerin SACompound 1.0 Triethanolamine 63.5 Aqua dem. A 10.0 CetearylEthylhexanoate 5.0 Tocopheryl Acetate 1.0 Bisabolol 0.1 Perfume 0.3Acrylates/C10-30 Alkyl Acrylate Crosspolymer B 15.0 Alcohol 1.0Panthenol 3.0 Glycerin SA Compound 0.1 Triethanolamine 59.5 Aqua dem.Production: Dissolve Phase A. Dissolve Phase B and add to Phase A.Homogenize by stirring.

AFTER SUN LOTIONS % Ingredients (INCI) A 0.4 Acrylates/C10-30 AlkylAcrylate Crosspolymer 15.0 Cetearyl Ethylhexanoate 0.2 Bisabolol 1.0Tocopheryl Acetate QS. Perfume B 1.0 Panthenol 15.0 Alcohol 3.0 GlycerinSA Compound 63.2 Aqua dem, C 0.2 Triethanolamine A 0.4 Acrylates/C10-30Alkyl Acrylate Crosspolymer 15.0 Cetearyl Ethylhexanoate 0.2 Bisabolol1.0 Tocopheryl Acetate QS Perfume B 1.0 Panthenol 15.0 Alcohol 3.0Glycerin SA Compound 59.2 Aqua dem, C 0.2 TriethanolamineProduction: Dissolve Phase A. Dissolve Phase B and add to Phase A.Homogenize by stirring. Neutralize Phase A and B by adding Phase C andhomogenize again.

SUNSCREEN LOTIONS % Ingredients (INCI) A 4.5 Ethylhexyl Methoxycinnamate2.0 Diethylamino Hydroxybenzoyl Hexyl Benzoate 3.0 Octocrylene 2.5Di-C12-13 Alkyl Malate 0.5 Tocopheryl Acetate 4.0 Polyglyceryl-3 MethylGlucose Distearate B 3.5 Cetearyl Isononanoate 1.0 VP/Eicosene Copolymer5.0 Isohexadecane 2.5 Di-C12-13 Alkyl Malate 3.0 Titanium Dioxide,Trimethoxycaprylylsilane C 5.0 Glycerin 1.0 Sodium Cetearyl Sulfate 0.5Xanthan Gum 59.7 Aqua dem. D SA Compound 1.0 Phenoxyethanol,Methylparaben, Ethylparaben, Butylparaben, Propylparaben,Isobutylparaben 0.3 Bisabolol A 4.5 Ethylhexyl Methoxycinnamate 2.0Diethylamino Hydroxybenzoyl Hexyl Benzoate 3.0 Octocrylene 2.5 Di-C12-13Alkyl Malate 0.5 Tocopheryl Acetate 4.0 Polyglyceryl-3 Methyl GlucoseDistearate B 3.5 Cetearyl Isononanoate 1.0 VP/Eicosene Copolymer 5.0Isohexadecane 2.5 Di-C12-13 Alkyl Malate 3.0 Titanium Dioxide,Trimethoxycaprylylsilane C 5.0 Glycerin 1.0 Sodium Cetearyl Sulfate 0.5Xanthan Gum 55.7 Aqua dem. D SA Compound 1.0 Phenoxyethanol,Methylparaben, Ethylparaben, Butylparaben, Propylparaben,Isobutylparaben 0.3 BisabololProduction: Heat Phase A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Heat Phase C to 80° C. and add tothe combined Phase A and B and homogenize again. Cool to approx. 40° C.and add Phase D. Homogenize again.

SUNSCREEN LOTIONS O/W % Ingredients (INCI) A 2.0 Ceteareth-6, StearylAlcohol 2.0 Ceteareth-25 3.0 Tribehenin 2.0 Cetearyl Alcohol 2.0Cetearyl Ethylhexanoate 5.0 Ethylhexyl Methoxycinnamate 1.0 EthylhexylTriazone 1.0 VP/Eicosene Copolymer 7.0 Isopropyl Myristate B 5.0 ZincOxide, Triethoxycaprylylsilane C 0.2 Xanthan Gum 0.5 HydroxyethylAcrylate/Sodium Acryloyldimethyl Taurate Copolymer, Squalane,Polysorbate 60 0.2 Disodium EDTA 5.0 Propylene Glycol 0.5 Panthenol 60.9Aqua dem. D SA Compound 0.5 Phenoxyethanol, Methylparaben, Butylparaben,Ethylparaben, Propylparaben, Isopropylparaben 1.0 Tocopheryl Acetate 0.2Bisabolol A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 3.0Tribehenin 2.0 Cetearyl Alcohol 2.0 Cetearyl Ethylhexanoate 5.0Ethylhexyl Methoxycinnamate 1.0 Ethylhexyl Triazone 1.0 VP/EicoseneCopolymer 7.0 Isopropyl Myristate B 5.0 Zinc Oxide,Triethoxycaprylylsilane C 0.2 Xanthan Gum 0.5 HydroxyethylAcrylate/Sodium Acryloyldimethyl Taurate Copolymer, Squalane,Polysorbate 60 0.2 Disodium EDTA 5.0 Propylene Glycol 0.5 Panthenol 56.9Aqua dem. D SA Compound 0.5 Phenoxyethanol, Methylparaben, Butylparaben,Ethylparaben, Propylparaben, Isopropylparaben 1.0 Tocopheryl Acetate 0.2BisabololProduction: Heat Phase A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Heat Phase C to 80° C. and add tothe combined Phase A and B and homogenize again. Cool to approx. 40° C.and add Phase D. Homogenize again.

SUNSCREEN LOTIONS O/W % Ingredients (INCI) A 3.5 Ceteareth-6, StearylAlcohol 1.5 Ceteareth-25 7.5 Ethylhexyl Methoxycinnamate 2.0Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Cyclopentasiloxane,Cyclohexasiloxane 0.5 Bees Wax 3.0 Cetearyl Alcohol 10.0 Caprylic/CapricTriglyceride B 5.0 Titanium Dioxide, Silica, Methicone, Alumina C 3.0Glycerin 0.2 Disodium EDTA 0.3 Xanthan Gum 1.0 Decyl Glucoside 2.0Panthenol, Propylene Glycol 56.3 Aqua dem. D SA Compound 1.0 TocopherylAcetate 0.2 Bisabolol QS Parfümöl QS Preservative A 3.5 Ceteareth-6,Stearyl Alcohol 1.5 Ceteareth-25 7.5 Ethylhexyl Methoxycinnamate 2.0Diethylamino Hydroxybenzoyl Hexyl Benzoate 2.0 Cyclopentasiloxane,Cyclohexasiloxane 0.5 Bees Wax 3.0 Cetearyl Alcohol 10.0 Caprylic/CapricTriglyceride B 5.0 Titanium Dioxide, Silica, Methicone, Alumina C 3.0Glycerin 0.2 Disodium EDTA 0.3 Xanthan Gum 1.0 Decyl Glucoside 2.0Panthenol, Propylene Glycol 52.3 Aqua dem. D SA Compound 1.0 TocopherylAcetate 0.2 Bisabolol QS Perfume QS PreservativeProduction: Heat Phase A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Heat Phase C to 80° C. and add tothe combined Phase A and B and homogenize again. Cool to approx. 40° C.and add Phase D. Homogenize again.

FOOT BALM % Ingredients (INCI) A 2.0 Ceteareth-6, Stearyl Alcohol 2.0Ceteareth-25 5.0 Cetearyl Ethylhexanoate 4.0 Cetyl Alcohol 4.0 GlycerylStearate 5.0 Mineral Oil 0.2 Menthol 0.5 Camphor B 69.3 Aqua dem. QSPreservative C 1.0 Bisabolol 1.0 Tocopheryl Acetate D SA Compound 5.0Witch Hazel Extract A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-255.0 Cetearyl Ethylhexanoate 4.0 Cetyl Alcohol 4.0 Glyceryl Stearate 5.0Mineral Oil 0.2 Menthol 0.5 Camphor B 65.3 Aqua dem. QS Preservative C1.0 Bisabolol 1.0 Tocopheryl Acetate D SA Compound 5.0 Witch HazelExtractProduction: Heat Phase A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Cool to approx. 40° C. and addPhase C and D. Homogenize by stirring and cool to room temperature

W/O % Ingredients (INCI) A 6.0 PEG-7 Hydrogenated Castor Oil 8.0Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 15.0 Mineral Oil 0.3Magnesium Stearate 0.3 Aluminum Stearate 2.0 PEG-45/Dodecyl GlycolCopolymer B 5.0 Glycerin 0.7 Magnesium Sulfate 55.6 Aqua dem. C 1.0Compound 0.5 Tocopheryl Acetate 0.6 Bisabolol A 6.0 PEG-7 HydrogenatedCastor Oil 8.0 Cetearyl Ethylhexanoate 5.0 Isopropyl Myristate 15.0Mineral Oil 0.3 Magnesium Stearate 0.3 Aluminum Stearate 2.0PEG-45/Dodecyl Glycol Copolymer B 5.0 Glycerin 0.7 Magnesium Sulfate51.6 Aqua dem. C 5.0 Compound 0.5 Tocopheryl AcetateProduction: Heat Phase A and B separately to approx. 85° C. Add Phase Bto Phase A and homogenize by stirring. Cool to approx. 40° C. and addPhase C. Homogenize by stirring and cool to room temperature.

LIQUID MAKE-UP - TYPE O/W % Ingredients (INCI) A 2.0 Ceteareth-6,Stearyl Alcohol 2.0 Ceteareth-25 6.0 Glyceryl Stearate 1.0 Cetyl Alcohol8.0 Mineral Oil 7.0 Cetearyl Ethylhexanoate 0.2 Dimethicone B 3.0Propylene Glycol 1.0 Panthenol QS Preservative 61.9  Aqua dem. C 0.1Bisabolol SA Compound QS Perfume D 5.7 C.I. 77 891, Titanium Dioxide 1.1Iron Oxides A 2.0 Ceteareth-6, Stearyl Alcohol 2.0 Ceteareth-25 6.0Glyceryl Stearate 1.0 Cetyl Alcohol 8.0 Mineral Oil 7.0 CetearylEthylhexanoate 0.2 Dimethicone B 3.0 Propylene Glycol 1.0 Panthenol QSPreservative 57.9  Aqua dem. C 0.1 Bisabolol SA Compound QS Perfume D5.7 C. I. 77 891, Titanium Dioxide 1.1 Iron OxidesProduction: Heat Phase A and B separately to approx. 80° C. Add Phase Bto Phase A and homogenize by stirring. Cool to 40° C. and add Phase Cand D. Homogenize again and cool to room temperature.

FOUNDATION Ingredient INCI Name % Water Phase Dow Corning 9011Cyclopentasiloxane, PEG-12 Dimethicone Elastomer Blend Copolymer 15.00Dow Corning 245 Cyclopentasiloxane 5.00 Fluid Silcare 31 M50 SV CaprylylTrimethicone 6.35 Propylparaben 0.05 AS 5811 Titanium Dioxide,Triethoxycaprylylsilane 7.50 AS 5131 Iron Oxides,Triethoxycaprylylsilane 0.70 AS 5146 Iron Oxides,Triethoxycaprylylsilane 0.05 AS 5126 Iron Oxides,Triethoxycaprylylsilane 0.35 AS 50230 Talc, Triethoxycaprylylsilane 3.50Oil Phase Deionized Water 53.30 1.80Butylene Glycol 6.00 Methylparaben0.20 Benzoic Acid 0.20 Compound SA

The pigments (AS 5811, 5131, 5146, 5126, and 50230; Color Techniques)and propylparaben are dispersed in Silcare 31 M50 SV (Clariant),stirring until wet. The mixture is then passed over a three roll mill attight setting until particle size is <10 μm. Then, DC 9011 ElastomerBlend (Dow Corning) and DC 245 Fluid are combined in finishing vessel,stirring until homogenous. The color grind is added with slowhomogenizer agitation. The water is weighed into a separate vessel andCompound is gradually added with propeller agitation, stirring untildissolved. Methylparaben and benzoic acid are added to butylene glycol.The mixture is warmed slightly, and stirred until dissolved. The mixtureis cooled to 30° C. and added to the Compound-containing solution. Thewater phase is added slowly to the oil phase with rapid agitation. Whenaddition is complete, the preparation is homogenized for five minutes.This preparation is useful as a makeup foundation for application toskin.

Mascara Formulation

The ingredients of both a control preparation and a mascara containing2% are as follows:

% % Trade Name INCI Name Control 1 Compound 2 Phase # Water Phase 1Deionized Water 42.96 42.96 2 Butylene Glycol 5.00 5.00 2 Methylparaben0.30 0.30 3 33-5198 (Black) Iron Oxides 10.00 10.00 (Sun) 4 Natrosol 250MR Hydroxyethylcellulose 0.20 0.20 (Aqualon) 5 10% KOH PotassiumHydroxide 0.01 0.01 6 Arlacel 165 Glyceryl Stearate, 3.00 3.00 PEG-100Stearate (Uniqema) 7 10% Citric Acid 0.27 0.27 Phase # Wax Phase 8Arlacel 165 1.00 1.00 8 Cerasynt SD Glyceryl Stearate (ISP) 3.50 3.50 8Beeswax, White Beeswax (S&P) 7.50 7.50 SP 424 8 Carnauba #1 CopernicaCerifera 4.80 4.80 (Carnauba) Wax (S&P) 8 Propylparaben 0.10 0.10 9Deionized Water 20.00 — 9 10% Compound/ — 20.00 Water 10 Deionized Water1.00 1.00 10 Glydant DMDM Hydantoin 0.36 0.36 (Lonza) 100.00 100.00

To produce the mascara formulation, the wax phase 8 is combined andheated to 85-90° C. with propeller mixing. The 10% Compound solution isprepared by adding Compound to water while propeller mixing. Phase 1water is added to a tared stainless steel beaker (approximately 50 gexcess is added to compensate for loss). Phase 2 methylparaben is addedto butylene glycol and stirred while warming on top of a steam bathuntil dissolved, then added to the water with slow homomixer agitation.Then, the phase 4 black iron oxide is added, while maintainingagitation. Then, Natrosol is sprinkled in, while maintaining agitation.The 10% KOH is added, and heating is begun to 85° C., with the beakercovered as tightly as possible. When the Natrosol is dissolved, the 10%citric acid is added dropwise, maintaining temperature and agitation.Then, the Arlacel 165 is added slowly and mixed for at least 5 minutesto insure dissolution. At 85-90° C., the wax phase is slowly added tothe water phase while homomixing. The temperature and agitation aremaintained for 10 minutes. The batch is removed from the steam bath andallowed to cool while homomixing with occasional hand scraping of thebeaker walls. At 55° C., the batch is weighed to check for water loss.Mixing is resumed and water is added back, if necessary. At 45° C.,phases 9 and 10 are added. Cooling is continued using cold water to 30°C. At this point, continuous hand scraping of beaker walls is necessary.

In this preparation, the small amount of KOH (in Phase 5) is used toraise the pH to disperse the Natrosol which is coated with glyoxal toretard wetting, and prevent agglomeration. In phase 7, the citric acidis added slowly to adjust pH to ˜5.5, below the isoelectric point of theiron oxides. In phases 7 and 8, the Arlacel 165 is split between the oiland water phases, as the emulsification is easier to accomplish withsurfactant in both phases. In phase 9, the deionized water is added inthe control batch instead of Compound. The Compound solution is preparedwhile the emulsion is being processed, so it is absolutely fresh. Thispreparation provides a formulation suitable for use as a mascara.

Example 18 Hair Growth Inhibition

In this Example, experiments to determine the ability of thecompositions of the present invention to inhibit the growth of hair aredescribed. In particular, these experiments are conducted in order toassess the ability of the compositions of the present invention todecrease hair growth after depilation by shaving or use of depilatorycreams or waxing.

A lotion for inhibiting hair growth and containing a modified variantBBPI in which the chymotrypsin loop of the parent BBPI is replaced witha VEGF-binding peptide is prepared according to the followingformulation (A):

Ingredient (INCI name) % in weight Water 85.97 Cetearyl alcohol 8.50Ceteareth-20 2.70 PPG-15 Stearyl Ether 0.60 Prunus Amygdalus Dulcis Oil(1) 0.10 Paraffinum Liquidum 0.10 Allantoin 0.20 Propylene glycol 0.13CI77891 Titanium dioxide 0.17 Tetra sodium EDTA 0.10, VEGF-BBPI 0.10Sodium Chloride 0.091 Citric acid 0.11 Sodium Hydroxyde 0.034Phenoxyethanol 1.0 Robertet Artlande G10029876 (2) 0.10

The composition includes at least one type of VEGF-BBPI chosen from SEQID NOS: 601, 602, 627-631, 643, 491, 632-636. In some embodiments, thecomposition comprises at least two, at least three, at least four or atleast five different types of VEGF-BBPIs chosen from SEQ ID NOS:601,602, 627-631, 643, 491, 632-636.

The formulation comprising the VEGF-BBPI is manufactured as follows:

1) Blending the fatty alcohol emulsifier and oil gelling agent togetherinto a molten phase at a temperature of 60, preferably 70° C. or more,

2) emulsifying the molten phase into an aqueous phase, the temperatureof the aqueous phase prior to emulsification being 50° C., preferably60° C., more preferably 70° C. or more, whereby an emulsion is formed,

3) cooling the emulsion to a temperature of 35° C. or less,

4) dispersing the perfume, preservative, citric acid solution buffersolution in the emulsion.

5) adding in the same manner the solution of VEGF-BBPI, finishing withthe buffer solution over a period of approximately 5 min.

6) Agitating the mixture for a further 10 minutes

A control formulation (B) is prepared according to the method describedfor the preparation of formulation A but excluding the VEGF-BBPI.

Facial Hair Experiments

In these experiments, a group (e.g., 5) male subjects with FitzpatrickSkin Classification II are tested. Individuals are requested to use notopical facial treatment prior to beginning the experiments. On day 1,facial hair growth is visually evaluated and photographed. Followingthis evaluation and photography, the composition(s) to be tested, aswell as a vehicle control are applied at the desired concentration(s).Beginning on day 2, the individuals apply the composition(s) immediatelyafter shaving. No other pre- or post-shave treatment is used for theduration of the experiments. In most cases, the experiment continues fora time period of 30 to 45 days. Facial hair growth is visually evaluatedand photographed every third day during the experiments. The number ofhairs, as well as the hair shaft length and width are measured usingcomputerized image analysis. In preferred embodiments, there is adecrease in the number of hairs, hair thickness and/or hair length dueto the application of the test compound(s).

Leg Hair Experiments

In these experiments, a group (e.g., 5) female subjects with FitzpatrickSkin Classification II are tested. Individuals are requested to use notopical leg treatment prior to beginning the experiments. On day 1,areas on both legs of each individual are marked and the hair growth isvisually evaluated and photographed. Following this evaluation andphotography, the composition(s) to be tested are applied at the desiredconcentration(s). Following this evaluation and photography, thecomposition(s) to be tested (i.e., test compounds containing a desiredconcentration of VEGF-BBP), as well as a vehicle control, are applied atthe desired concentration(s). In some methods, each individual isprovided with two tubes, one of which contains the VEGF-BBPI and theother containing the vehicle control. These tubes are marked “left” and“right.” Each day during the experiments, the subject applies thecompositions in the two tubes the respective legs. After 7 days ofapplication, the individuals are visually evaluated and photographs aretaken. Both legs are then shaved or exposed to a depilatory and the testindividuals continue to apply the compositions as before. Hair growth isthen evaluated visually and by photographing appropriate areas on thelegs every 2 days. After 10 days, the legs are again shaved and the testsubjects continue to apply the compositions as before. In some methods,the experiments are conducted for 3 cycles and the hair growth isvisually evaluated and photographs were taken. The experiments are thencontinued for an additional 8 days. In preferred embodiments, there is adecrease in the number of hairs, hair thickness and/or hair length dueto the application of the test compound(s) in the marked area(s).

Beginning on day 2, the individuals apply the composition(s) immediatelyafter shaving. No other pre- or post-shave treatment is used for theduration of the experiments. In most cases, the experiment continues fora time period of 30 to 45 days. Facial hair growth is visually evaluatedand photographed every third day during the experiments. The number ofhairs, as well as the hair shaft length and width are measured usingcomputerized image analysis. In preferred embodiments, there is adecrease in the number of hairs, hair thickness and/or hair length dueto the application of the test compound(s).

1. An isolated modified variant Bowman Birk Protease Inhibitor (BBPI)comprising a BBPI scaffold selected from the group consisting of: BBI(SEQ ID NO: 13), BBIt (SEQ ID NO:185), BBI-AV (SEQ ID NO:186), BBIt-AV(SEQ ID NO:187), BBIt-VEGK (SEQ ID NO:640), BBIt-VEGT (SEQ ID NO:641),BBIt-VEGKD (SEQ ID NO:642), BBdb (SEQ ID NO:449), BBsb3 (SEQ ID NO:450),BBtc (SEQ ID NO:451), BBdb-AV (SEQ ID NO:452), BBsb3-AV (SEQ ID NO:453)and BBtc-AV (SEQ ID NO:454), wherein the second protease inhibitory loopof the BBPI scaffold is replaced with a binding peptide selected fromthe group consisting of a vascular endothelial growth factor (VEGF)binding peptide, a fibroblast growth factor-5 (FGF-5) binding peptide, atransforming growth factor β (TGFβ) binding peptide and a tumor necrosisfactor α (TNFα) binding peptide; and wherein the backbone of the BBPIscaffold is substituted at one or more amino acid positions selectedfrom the group consisting of positions equivalent to positions 1, 4, 5,11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55, and 65 of SEQ ID NO:187;wherein the substitution at position 1 is to A or C, the substitution atposition 4 is to V, the substitution at position 5 is to P or A, thesubstitution at position 11 is to G, the substitution at position 13 isto Y, I, F, M, L, V, K or R, the substitution at position 18 is to I, Vor L, the substitution at position 25 is to K, N, W, I, A, R or L, thesubstitution at position 27 is to R, K, V, A or Q, the substitution atposition 29 is to R, K, P or E, the substitution at position 31 is to Q,H, E, A, R, W, K or T, the substitution at position 38 is to N, K or R,the substitution at position 40 is to H, K, Q, R or Y, the substitutionat position 50 is to R, Q, K, T, V, M or S, the substitution at position52 is to K, T, R, Q, L, H, A, M, S or E, the substitution at position 55is to M and the substitution at position 65 is to E, Q or D.
 2. Theisolated modified variant BBPI of claim 1, wherein said binding peptideis a VEGF binding peptide chosen from YNLYGWT (SEQ ID NO: 676),ACYNLYGWTC (SEQ ID NO:9), KYYLYWW (SEQ ID NO:458), TLWKSYW (SEQ IDNO:459), DLYWW (SEQ ID NO:460),SKHSQIT (SEQ ID NO:468) KTNPSGS (SEQ IDNO:469) RPTGHSL (SEQ ID NO:470), KHSAKAE (SEQ ID NO:471) KPSSASS (SEQ IDNO:472), PVTKRVH (SEQ ID NO:473), TLHWWVT (SEQ ID NO:492), PYKASFY (SEQID NO:493), PLRTSHT (SEQ ID NO:494), EATPROT (SEQ ID NO:495), NPLHTLS(SEQ ID NO:496), KHERIWS (SEQ ID NO:497), ATNPPPM (SEQ ID NO:498),STTSPNM (SEQ ID NO:499), ADRSFRY (SEQ ID NO:500), PKADSKQ (SEQ IDNO:501), PNQSHLH (SEQ ID NO:502), SGSETWM (SEQ ID NO:503), ALSAPYS (SEQID NO:504), KMPTSKV (SEQ ID NO:505), ITPKRPY (SEQ ID NO:506), KWIVSET(SEQ ID NO:507), PNANAPS (SEQ ID NO:508), NVQSLPL (SEQ ID NO:509),TLWPTFW (SEQ ID NO:510), NLWPHFW (SEQ ID NO:511), SLWPAFW (SEQ IDNO:512), SLWPHFW (SEQ ID NO:513), APWNSHI (SEQ ID NO:514), APWNLHI (SEQID NO:515), LPSWHLR (SEQ ID NO:516), PTILEWY (SEQ ID NO:517), TLYPQFW(SEQ ID NO:518), HLAPSAV (SEQ ID NO:519), KYYLSWW (SEQ ID NO:520),WYTLYKW (SEQ ID NO:521), TYRLYWW (SEQ ID NO:522), RYSLYYW (SEQ IDNO:523), YYLYYWK (SEQ ID NO:524), NYQLYGW (SEQ ID NO:525), TKWPSYW (SEQID NO:226), TLWKSYW (SEQ ID NO:527), PLWPSYW (SEQ ID NO:528), RLWPSYW(SEQ ID NO:529), TLWPKYW (SEQ ID NO:530), KYDLYWW (SEQ ID NO: 531),RYDLYWW (SEQ ID NO:532), DYRLYWW (SEQ ID NO:533), DYKLYWW (SEQ IDNO:534), EYKLYWW (SEQ ID NO:535), and RYPLYWW (SEQ ID NO:536).
 3. Theisolated modified variant BBPI of claim 1, wherein said binding peptideis an FGF-5 binding peptide chosen from CACRTQPYPLCF (MM007; SEQ IDNO:430), CICTWIDSTPC (PS2; SEQ ID NO:431), CYGLPFTRC (SEQ ID NO:537),CEEIWTMLC (SEQ ID NO:538), CWALTVKTC (SEQ ID NO:539), CLTVLWTTC (SEQ IDNO:540), CTLWNRSPC (SEQ ID NO:541), CHYLLTNYC (SEQ ID NO:542), CRIHLAHKC(SEQ ID NO:543), TNIDSTP(SEQ ID NO:544), HLQTTET (SEQ ID NO:545),SLNNLTV (SEQ ID NO:546), TNIDSTP (SEQ ID NO:547), TNIDSTP (SEQ IDNO:548), LRILANK (SEQ ID NO:549), LLTPTLN (SEQ ID NO:550), ALPTHSN (SEQID NO:551), TNIDSTP (SEQ ID NO:552), LCRRFEN (SEQ ID NO:553), TNIDSTP(SEQ ID NO:554), TNIDSTP (SEQ ID NO:555), HLQTTET (SEQ ID NO:556),PLGLCPP (SEQ ID NO:557), GYFIPSI (SEQ ID NO:558), TKIDSTP (SEQ IDNO:559), HLQTTET (SEQ ID NO:560), WNIDSTP (SEQ ID NO:561), TWIDWTP (SEQID NO:562), RTQPYPL (SEQ ID NO:670) and TWIDSTP (SEQ ID NO:671).
 4. Theisolated modified variant BBPI of claim 1, wherein said binding peptideis a TGFβ binding peptide chosen from CLCPENINVLPCN (PEN3; SEQ IDNO:436), CICKHNVDWLCF (MMO21W; SEQ ID NO:437), CICWTQHIHNCF (WTQ; SEQ IDNO:438), CVTTDWIEC (SEQ ID NO:563), CYYSQFHQC (SEQ ID NO:564), CPTLWTHMC(SEQ ID NO:565), QSACIVYYVGRKPKVECASSD (SEQ ID NO:566),QSACILYYIGKTPKIECASSD (SEQ ID NO:567), QSACILYYVGRTPKVECASSD (SEQ IDNO:568), KHNVRLL (SEQ ID NO:570), NDTPSYF (SEQ ID NO:571), AKLYAGS (SEQID NO:572), RGPAHSL (SEQ ID NO:573), NSLAERR (SEQ ID NO:574), HPLASPH(SEQ ID NO:575), QPWNKLK (SEQ ID NO:576), PTKPAQQ (SEQ ID NO:578),PSLNRPQ (SEQ ID NO:579), HHARQEW (SEQ ID NO:580), RHHTPGP (SEQ IDNO:581), ASAINPH (SEQ ID NO:582), CHGYDRAPC (SEQ ID NO:644), CFAPADQAC(SEQ ID NO:645), CIPSRFITC (SEQ ID NO:646), CHGHTKLAC (SEQ ID NO:647),CNGKSKLAC (SEQ ID NO:648), PENINVLP (SEQ ID NO:672), KHNVDWL (SEQ IDNO:673) and WTQHIHNC (SEQ ID NO:674).
 5. The isolated modified variantBBPI of claim 1, wherein said binding peptide is a TNFα binding peptidechosen from RYWQDIP (T1; SEQ ID NO:474), APEPILA (T2; SEQ ID NO:475),DMIMVSI (T3; SEQ ID NO:476), WTPKPTQ (SEQ ID NO:583), ATFPNQS (SEQ IDNO:584), ASTVGGL (SEQ ID NO:585), TMLPYRP (SEQ ID NO:586), AWHSPSV (SEQID NO:587), TQSFSS (SEQ ID NO:588), THKNTLR (SEQ ID NO:589), GQTHFHV(SEQ ID NO:590), LPILTQT (SEQ ID NO:591), SILPVSH (SEQ ID NO:592),SQPIPI (SEQ ID NO:593), and QPLRKLP (SEQ ID NO:594).
 6. The isolatedmodified variant BBPI of claim 1, wherein said modified variant BBPI isexpressed as a fusion protein comprising a catalytic domain chosen fromcellulase, cutinase and disulfide isomerase.
 7. The isolated modifiedvariant BBPI of claim 1, wherein said modified variant BBPI is expressedas a fusion protein consisting of SEQ ID NO:195.
 8. The isolatedmodified variant BBPI of claim 1, wherein the backbone of the BBPIscaffold is substituted at a combination of two amino acid positionsequivalent to positions 50 and 52 of SEQ ID NO:187.
 9. The isolatedmodified variant BBPI of claim 8, wherein said modified variant BBPIcomprises SEQ ID NO:595.
 10. The isolated modified variant BBPI of claim1, wherein the backbone of the BBPI scaffold is substituted at acombination of three amino acid positions chosen from amino acidpositions equivalent to positions 25, 29, 40, 50 and 52 of SEQ IDNO:187.
 11. The isolated modified variant BBPI of claim 10, wherein saidcombination is chosen from 25L-50T-52A, 29P-50T-52A, and 40K-50T-52A.12. The isolated modified variant BBPI of claim 10, wherein saidmodified variant BBPI comprises SEQ ID NO:603, 607 or
 609. 13. Theisolated modified variant BBPI of claim 1, wherein the backbone of theBBPI scaffold is substituted at a combination of four amino acidpositions chosen from amino acid positions equivalent to positions 13,25, 29, 40, 50 and 52 of SEQ ID NO:187.
 14. The isolated modified BBPIof claim 13, wherein said combination is chosen from 13I-25L-50T-52A,13I-29P-50T-52A, 13I-40K-50T-52A, 25L-29P-50T-52A, 25L-40K-50T-52A,29P-40K-50T-52A and 13I-25K-29P-52K.
 15. The isolated modified variantBBPI of claim 14, wherein said modified variant BBPI comprises SEQ IDNO: 432, 434 443, 445, 447, 596, 600, 602, 604, 606, 608, or
 643. 16.The isolated modified variant BBPI of claim 1, wherein the backbone ofthe BBPI scaffold is substituted at a combination of five amino acidpositions selected from amino acid positions equivalent to positions 13,25, 29, 40, 50, and 52 of SEQ ID NO:187.
 17. The isolated modifiedvariant BBPI of claim 16, wherein said combination is chosen from13I-25L-29P-50T-52A, 13I-25L-40K-50T-52A, 13I-29P-40K-50T-52A,25L-29P-40K-50T-52A, 13I-29P-40K-50K-52A, and 13I-29P-40K-50T-52T. 18.The isolated modified variant BBPI of claim 17, wherein said modifiedvariant BBPI comprises SEQ ID NO: 597, 599, 601, 605, 615, 620, 624, or625.
 19. The isolated modified variant BBPI of claim 1, wherein thebackbone of the BBPI scaffold is substituted at a combination of sixamino acid positions chosen from amino acid positions equivalent topositions 1, 4, 5, 11, 13, 25, 27, 29, 31, 38, 40, 50, 52 and 65 of SEQID NO:187.
 20. The isolated modified variant BBPI of claim 19, whereinsaid combination is chosen from 13I-25L-29P-40K-50T-52A,1C-13I-29P-40K-50T-52A, 4V-13I-29P-40K-50T-52A, 5P-13I-29P-40K-50T-52A,11G-13I-29P-40K-50T-52A, 13I-25R-29P-40K-50T-52A,13I-27R-29P-40K-50T-52A, 13I-29P-31A-40K-50T-52A,13I-29P-31R-40K-50T-52A, 13I-29P-38N-40K-50T-52A, and13I-29P-40K-50T-52A-65E.
 21. The isolated modified variant BBPI of claim20, wherein said modified variant BBPI comprises SEQ ID NOS:598, 611,612, 613, 614, 616, 619, 621, 622, 623, or
 626. 22. The isolatedmodified variant BBPI of claim 1, wherein the backbone of the BBPIscaffold is substituted at a combination of seven amino acid positionsequivalent to positions 13, 25, 29, 31, 40, 50 and 52 of SEQ ID NO:187.23. The isolated modified variant BBPI of claim 22, wherein saidcombination is chosen from 13L-25R-29P-31A-40K-50T-52A,13L-25R-29P-31R-40K-50T-52A and 13I-25R-27A-29P-31A-50K-52T.
 24. Theisolated modified variant BBPI of claim 22, wherein said modifiedvariant BBPI comprises SEQ ID NOS: 491, 617, 618, 632, 633, 634, 635,636, 637, 638 or
 639. 25. The isolated modified variant BBPI of claim 1,wherein the backbone of the BBPI scaffold is substituted at acombination of eight amino acid positions equivalent to positions 13,25, 27, 29, 31, 40, 50 and 52 of SEQ ID NO:187.
 26. The isolatedmodified BBPI of claim 25, wherein said combination is chosen from13I-25R-27A-29P-31A-40H-50K-52T, 13I-25K-27A-29R-31E-40K-50Q-52Q,13I-25K-27R-29E-31A-40H-50R-52K, 13I-25K-27A-29R-31A-40H-50R-52L and13I-25K-27Q-29P-31E-40H-50R-52Q.
 27. The isolated modified variant BBPIof claim 25, wherein said modified variant BBPI comprises SEQ ID NO:627, 628, 629, 630 or
 631. 28. The isolated modified variant BBPI ofclaim 1, wherein said modified variant BBPI binds VEGF.
 29. The isolatedmodified variant BBPI of claim 1, wherein said VEGF binding peptide ischosen from SEQ ID NOS: 676, 9, 458, 459, 460, 468, 469, 470, 471, 472and
 473. 30. The isolated modified variant BBPI of claim 1, wherein saidmodified variant BBPI binds VEGF and comprises SEQ ID NO: 601, 602, 627,628, 630, 631, 643, 491, 632, 633, 634, 635, or
 636. 31. The isolatedmodified variant BBPI of claim 1, wherein the substitutions in thebackbone of the BBPI scaffold increase the trypsin inhibitory activityof the modified variant BBPI.
 32. The isolated modified variant BBPI ofclaim 1, wherein the substitutions in the backbone of the BBPI scaffoldincrease the trypsin inhibitory activity and production yield of themodified variant BBPI.
 33. An isolated modified variant Bowman BirkProtease Inhibitor (BBPI) comprising a BBPI scaffold consisting ofBBIt-AV (SEQ ID NO:187), wherein the VEGF binding peptide of the BBPIscaffold is replaced with a binding peptide selected from the groupconsisting of a FGF-5 binding peptide, a TGFβ binding peptide and a TNFαbinding peptide; and wherein the backbone of the BBPI scaffold issubstituted at one or more amino acid positions selected from the groupconsisting of 13, 25, 27, 29, 31,40, 50 and 52; wherein the substitutionat position 13 is to Y, I, F, M, L, V, K or R, the substitution atposition 25 is to K, N, W, I, A or R, the substitution at position 27 isto R, K, V, A or Q, the substitution at position 29 is to R, K or P, thesubstitution at position 31 is to Q, H, E, A, R, W, K or T, thesubstitution at position 40 is to H, K, Q, R or Y, the substitution atposition 50 is to R, Q, K, T, V, M or S, the substitution at position 52is to K, T, R, Q, L, H, A, M, S or E.
 34. The isolated modified variantBBPI of claim 33, wherein said binding peptide is an FGF binding peptidechosen from SEQ ID NOS: 430, 431, 670, and
 671. 35. The isolatedmodified variant BBPI of claim 33, wherein said binding peptide is a TGFbinding peptide chosen from SEQ ID NOS: 436, 437, 438, 672, 673, and674.
 36. The isolated modified variant BBPI of claim 33, wherein saidbinding peptide is a TNF binding peptide chosen from SEQ ID NOS: 474,475, and
 476. 37. The isolated modified variant BBPI of claim 33,wherein the substitutions in the backbone of the BBPI scaffold increasethe trypsin inhibitory activity and production yield of the modifiedvariant BBPI.
 38. The isolated modified variant BBPI of claim 33,wherein said modified variant BBPI comprises SEQ ID NO: 432, 434, 443,445, 447, 637, 638, and 639.